Ketol-acid reductoisomerase enzymes and methods of use

ABSTRACT

Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims the benefit of priority toU.S. Provisional Patent Application No. 61/645,832 filed on May 11, 2012and U.S. Patent Application No. 61/787,480 filed on Mar. 15, 2013, bothof which are herein incorporated by reference in their entireties.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under AgreementDE-AR0000006 awarded by the United States Department of Energy. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to polypeptides comprising ketol-acidreductoisomerase activity, polynucleotides encoding such polypeptides,host cells comprising such polynucleotides and polypeptides, and methodsof using such compositions.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the Sequence Listing submitted electronically herewith(Name: CL5631USNP_SEQLIST.txt; Size: 1,036,487 bytes; Date of Creation:Sep. 18, 2013) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Ketol-acid reductoisomerase (KARI) enzymes are involved in thebiological production of valine and isoleucine. KARI enzymes have alsobeen shown to be useful for pathways for the production of isobutanolusing engineered microorganisms (U.S. Pat. Nos. 7,851,188 and7,993,889). Such microorganisms can be used to produce isobutanol fromplant-derived substrates.

While methods for the chemical synthesis of isobutanol are known (oxosynthesis, catalytic hydrogenation of carbon monoxide (Ullmann'sEncyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbetcondensation of methanol with n-propanol (Carlini et al., J. Molec.Catal. A. Chem. 220215-220, 2004)), these processes use startingmaterials derived from petrochemicals and are generally expensive.Furthermore, chemical synthesis of isobutanol does not have thepotential for environmental advantages such as minimization of greenhouse gas emissions. Production of isobutanol from plant-derived rawmaterials would represent an advance in the art.

A KARI enzyme that can utilize reduced nicotinamide adenine dinucleotide(NADH) can capitalize on the NADH produced by the existing glycolyticpathway and other metabolic pathways in commonly used microbial cellsand may result in improved isobutanol production. U.S. Pat. No.8,129,162 and US Appl. Pub. No. 2010/0197519A1 describe the generationof KARI enzymes with varying abilities to utilize the cofactor (NADH).However, there remains a need in the art for alternative polypeptidesthat have KARI activity suitable for production pathways such asisobutanol biosynthetic pathways.

SUMMARY OF THE INVENTION

Provided herein is a polypeptide having ketol-acid reductoisomeraseactivity, wherein the amino acid at the position corresponding to 52 ofSEQ ID NO: 2 is D or E, and wherein the polypeptide comprises at leastone substitution in one of the following regions: in at least one of theinter-molecular dimer interface region, inter-domain interface region,N-domain surface helix region, hydrophobic bridge region, or theC-terminal tail region, wherein the at least one substitution is at theposition corresponding to I13, V53, A68, A69, A71, G72, V76, S86, L88,K99, Y113, N114, V117, I127, I152, S157, V171, M238, Y239, E264, N267,A268, Q272, R275, A277, R280, Y286, I291, S292, A295, M301, R306, S322,A329, K335, or A336 of SEQ ID NO: 2. In embodiments, the polypeptidefurther comprises a substitution at at least one of position L33, P47,F50, F61, I80, or V156. In embodiments, the polypeptide comprises atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99% identity to SEQ ID NO: 2

Provided herein are polypeptide variants comprising at least about 80%identity, at least about 85%, at least about 90%, at least about 95%, orat least about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2 and asubstitution at at least one of the following positions: Y286, I152,S322, S292, I291, I13, P47, F50, V53, A268, V76, A336, L88, A71, G72,M301, Y239, Y113, N114, A329, A69, A295, E264, R280, S157, M238, Q272,K335, K99, R275, or R306. In embodiments, the polypeptides haveketol-acid reductoisomerase activity.

Accordingly, provided herein are polypeptides comprising (a) the aminoacid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 116, 118, 119, 120, 121, 123, 124, 128, 129,131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 159, 160, 161, 346,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,378, 379, 380, 381, or 382; (b) at least about 95% or at least about 99%identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 116, 118, 119, 120, 121, 123, 124, 128, 129, 131,132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 157, 159, 160, 161, 346, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, or 382; or (c) an active fragment of (a) or (b). In someembodiments, microbial host cells comprise such polypeptides. In someembodiments, such microbial host cells produce isobutanol.

Polypeptides provided herein include polypeptides having ketol-acidreductoisomerase activity and comprising at least about 85%, at leastabout 95%, or at least about 99% identity to SEQ ID NO: 2 and at leastone of the following substitutions: Y286F, A336R, I152V, S322A, S292A,I291V, or a combination thereof; or an active fragment of suchpolypeptide. In embodiments, the polypeptides further comprise at leastone of the following substitutions: I13L, P47Y, F50A, V53A, A268E, V761,A336G, L88V, or a combination thereof. In embodiments, polypeptidescomprise 95% or at least about 99% identity to SEQ ID NO: 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27,28, or 29.

Polypeptides provided herein include polypeptides having ketol-acidreductoisomerase activity and comprising at least about 85%, at leastabout 95%, or at least about 99% identity to SEQ ID NO: 2 and at leastone of the following substitutions: Y286F, A71K, G72W, A336R, I152V,A268T, A329E, or a combination thereof; or an active fragment of suchpolypeptide. In embodiments, polypeptides comprise at least about 95% orat least about 99% identity to SEQ ID NO: 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, or 64.

Polypeptides provided herein include polypeptides having ketol-acidreductoisomerase activity and comprising at least about 85%, at leastabout 95%, or at least about 99% identity to SEQ ID NO: 41: and at leastone of the following substitutions M301I, Y239H, Y113F, S322A, A71K,N114G, A329R, A69T, N114S, G72W, A295V, E264K, R280D, A329E, S157T,M238I, Q272T, K335M, R280H, or a combination thereof; or an activefragment of such polypeptide. In embodiments, polypeptides comprise atleast about 95% or at least about 99% identity to SEQ ID NO: 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,116, 118, 119, 120, 121, 123, 124, 128, 129, 131, 132, 133, 134, 136,137, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 157, 159, 160, or 161. In embodiments, thepolypeptide having ketol-acid reductoisomerase activity comprises theamino acid sequence of SEQ ID NO: 365, 366, 159, 152, 377, 367, 123, 42,or 41.

Polypeptides provided herein include polypeptides having ketol-acidreductoisomerase activity and comprising at least about 85%, at leastabout 95%, or at least about 99% identity to SEQ ID NO: 346 and at leastone of the following substitutions: A68E, I152V, Y286F, and A336R.

The polypeptides provided here also may increase biosynthetic pathwayproductivity as compared to SEQ ID NO:2 or wild type ketol-acidreductoisomerase enzymes. The increase in productivity may include anincrease in performance of a step of the pathway such as rate of thereaction catalyzed by the ketol-acid reductoisomerase and/or reducedsusceptibility to inhibition or substrate competition. The increase inproductivity may also manifest in an increase in product yield such asincreased isobutanol production as compared to polypeptides without themodifications described herein such as SEQ ID NO: 2. The productivityincrease may be on the order of at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% to about 100%.

In embodiments, in polypeptides provided herein, the amino acid at theposition corresponding to position 52 of SEQ ID NO: 2 is D or E. Inembodiments, the amino acid at the position corresponding to position 24of SEQ ID NO: 2 is F; the amino acid at the position corresponding toposition 33 of SEQ ID NO: 2 is L; the amino acid at the positioncorresponding to position 47 of SEQ ID NO: 2 is P; the amino acid at theposition corresponding to position 50 of SEQ ID NO: 2 is F; the aminoacid at the position corresponding to position 61 of SEQ ID NO: 2 is F;the amino acid at the position corresponding to position 80 of SEQ IDNO: 2 is I; and the amino acid at the position corresponding to position156 of SEQ ID NO: 2 is V.

Also provided herein are polynucleotides encoding such polypeptides.Also provided are recombinant microbial host cell comprising suchpolynucleotides. Also provided herein are recombinant microbial hostcells comprising such polypeptides. In some embodiments, microbial hostcells further comprise reduced or eliminated acetolactate reductaseactivity. In embodiments, host cells further comprises at least onedeletion, mutation, and/or substitution in fra2. In embodiments, hostcells further comprise the substrate to product conversions: pyruvate toacetolactate; acetolactate to 2,3-dihydroxyisovalerate;2,3-dihydroxyisovalerate to α-ketoisovalerate; α-ketoisovalerate toisobutyraldehyde; and isobutyraldehyde to isobutanol. In embodiments,the polypeptide having ketol-acid reductoisomerase activity is selectedfrom the group consisting of SEQ ID Nos: 11, 41, and 42. In embodiments,the polypeptide having ketol-acid reductoisomerase activity comprisesthe amino acid sequence of SEQ ID NO: 365, 366, 159, 152, 377, 367, 123,42, or 41. In embodiments, the host cell is a yeast host cell. Inembodiments, the host cell is Saccharomyces cerevisiae.

Also provided herein are methods for converting acetolactate todihydroxyisovalerate comprising the polypeptides provided. Also providedare methods for converting acetolactate to dihydroxyisovaleratecomprising providing a microbial host cell comprising a polypeptideprovided; and contacting the polypeptide with acetolactate whereindihydroxyisovalerate is produced. Also provided are methods of producinga product selected from the group consisting of isobutanol,pantothenate, valine, leucine, isoleucine, 3,3-dimethylmalate, and2-methyl-1-butanol comprising: providing a recombinant host cellprovided herein wherein the recombinant host cell comprises a productbiosynthetic pathway; and contacting the microbial host cell with acarbon substrate under conditions whereby the product is produced. Inembodiments, at least a portion of the contacting occurs under anaerobicconditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-Shows four different isobutanol biosynthetic pathways. The stepslabeled “a”, “b”, “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j” and “k”represent the substrate to product conversions described below.

FIG. 2 depicts an alignment of the amino acid sequences of the KARI fromPseudomonas fluorescens (“PF5”; SEQ ID NO: 1) and a variant thereof(“JEA1”; SEQ ID NO: 2; described in U.S. Appn. Pub. No. 2010/0197519which is incorporated herein by reference).

FIG. 3 is a plasmid map of pLH556 (pHR81-PIlv5-Pf5.KARI) (SEQ ID NO:162). Plasmid pLH556 contains a 2-micron origin of replication foryeast, a pBR322 origin for E. coli, Leu2 and Ura3 auxotrophic markergenes for maintenance in yeast, an ampicillin resistance marker gene formaintenance in E. coli, and a KARI open reading frame cloned betweenPmeI and Sfil restriction sites. The KARI promoter is ilv5p and the KARIterminator is ilv5t.

FIG. 4 is a plasmid map of pBP915 (SEQ ID NO: 163). Plasmid pBP915contains a 2-micron origin of replication for yeast, a pMB1 origin forE. coli, and an F1 origin for phage F1. It contains a His3 auxotrophicmarker gene for maintenance in yeast, and an ampicillin resistancemarker gene for maintenance in E. coli. The IlvD (DHAD) gene from S.mutans strain UA159 is cloned between AfII and NotI restriction sites.The ilvD promoter is FBAp and the ilvD terminator is FBAt. The horsealcohol dehydrogenase gene is also present, with a GPMp promoter andADHt terminator.

FIG. 5 is a plasmid map of pBP2090 (pHR81-PIIv5p.K9D3.TEF1(M4)p.ilvD)vector (SEQ ID NO: 164). Plasmid pBP2090 contains a 2-micron origin ofreplication for yeast, a pBR322 origin for E. coli, Leu2 and Ura3auxotrophic marker genes for maintenance in yeast, an ampicillinresistance marker gene for maintenance in E. coli, and a KARI ORF clonedbetween PmeI and SfiI restriction sites. The KARI promoter is ilv5p andthe KARI terminator is ilv5t. The plasmid also contains the IIvD (DHAD)gene from S. mutans strain UA159 cloned between AfIII and NotIrestriction sites. The ilvD promoter is a modified TEF1 promoter(TEF1(M4)p) and the ilvD terminator is FBAt.

FIG. 6 shows an isobutanol biosynthetic pathway. Step “a” represents theconversion of pyruvate to acetolactate. Step “b” represents theconversion of acetolactate to DHIV. Step “c” represents the conversionof dihydroxyisovalerate (DHIV) to ketoisovalerate (KIV). Step “d”represents the conversion of KIV to isobutyraldehyde. Step “e”represents the conversion of isobutyraldehyde to isobutanol. Step “f”represents the conversion of acetolactate to2,3-dihydroxy-2-methylbutyrate (DHMB).

FIG. 7 is a plasmid map of an example of the vector (SEQ ID NO: 347;containing the coding sequence for KARI variant 65139) used incombination with the plasmid given as SEQ ID NO: 163 in the Exampleswhere indicated. This plasmid is similar to the plasmid depicted in FIG.3, but the Ura3 gene has been modified with a silent mutation toeliminate the BsmBI site within.

FIG. 8 is a plasmid map of an example of the vector (SEQ ID NO: 349;containing the coding sequence for KARI variant 76437 “R9E8”) used inthe Examples where indicated. This plasmid is similar to the plasmiddepicted in FIG. 5, but the Ura3 gene has been modified with a silentmutation to eliminate the BsmBI site within.

FIG. 9 shows the homology model of Pf5 KARI dimer. Positions I13, I152and S157 are indicated (arrows) in one of the monomers.

FIGS. 10A and 10B show the homology model of Pf5 KARI, monomer A only.The N-domain (residues 1-181) and the C-domain (residues 182-327) areshown. In FIG. 10A, the location of positions M238, Y239, E264, N267,A268, Q272, A277, R280, Y286, I291, S292, M301, S322, in theinter-molecular dimer interface is shown. In FIG. 10B, the location ofpositions Y113, N114, N114, I291, S292, and A295, in the inter-domaininterface region are shown.

FIG. 11A shows the positions of P47, P50, V53, T80, and L88, part of themodeled nucleotide binding region. FIG. 11B shows positions 68, 69, 71and 72 on an alpha helix of two short turns (positions 64-73) on thesurface of the N-domain. Residue 76 in the model interacts with the sidechain of residue 70 through hydrophobic interaction. The N-terminalportion of this short helix is packed against the main chain of residue47 part of the modeled nucleotide binding region.

FIG. 12 shows the positions C33, L61, I127, I152, and V171 in theN-domain hydrophobic bridge region.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference, unless only specific sections of patents orpatent publications are indicated to be incorporated by reference.

Although methods and materials similar or equivalent to those disclosedherein can be used in practice or testing of the present invention,suitable methods and materials are disclosed below. The materials,methods and examples are illustrative only and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

In order to further define this invention, the following terms,abbreviations and definitions are provided.

It will be understood that “derived from” with reference to polypeptidesdisclosed herein encompasses sequences synthesized based on the aminoacid sequences of the KARIs present in the indicated organisms as wellas those cloned directly from the organism's genetic material.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers and are intended to be non-exclusive or open-ended.For example, a composition, a mixture, a process, a method, an article,or an apparatus that comprises a list of elements is not necessarilylimited to only those elements but can include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers can be added to thespecified method, structure, or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition. See M.P.E.P. §2111.03.

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances, i.e., occurrences of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the claims as presented or as later amended andsupplemented, or in the specification.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates orsolutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or to carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, or within 5% of the reported numerical value.

The term “isobutanol biosynthetic pathway” refers to the enzymaticpathway to produce isobutanol. For example, certain isobutanolbiosynthetic pathways are disclosed in U.S. Pat. No. 7,851,188, which isincorporated by reference in its entirety herein. Certain isobutanolbiosynthetic pathways are illustrated in FIG. 1 and described herein.From time to time “isobutanol biosynthetic pathway” is used synonymouslywith “isobutanol production pathway”.

The term “butanol” as used herein refers to 2-butanol, 1-butanol,isobutanol or mixtures thereof. Isobutanol is also known as2-methyl-1-propanol.

A recombinant host cell comprising an “engineered alcohol productionpathway” (such as an engineered butanol or isobutanol productionpathway) refers to a host cell containing a modified pathway thatproduces alcohol in a manner different than that normally present in thehost cell. Such differences include production of an alcohol nottypically produced by the host cell, or increased or more efficientproduction.

The term “heterologous biosynthetic pathway” as used herein refers to anenzyme pathway to produce a product in which at least one of the enzymesis not endogenous to the host cell containing the biosynthetic pathway.

The term “extractant” as used herein refers to one or more solventswhich can be used to extract butanol from a fermentation broth.

The term “effective titer” as used herein, refers to the total amount ofa particular alcohol (e.g. butanol) produced by fermentation per literof fermentation medium. The total amount of butanol includes: (i) theamount of butanol in the fermentation medium; (ii) the amount of butanolrecovered from the organic extractant; and (iii) the amount of butanolrecovered from the gas phase, if gas stripping is used.

The term “effective rate” as used herein, refers to the total amount ofbutanol produced by fermentation per liter of fermentation medium perhour of fermentation.

The term “effective yield” as used herein, refers to the amount ofbutanol produced per unit of fermentable carbon substrate consumed bythe biocatalyst.

The term “separation” as used herein is synonymous with “recovery” andrefers to removing a chemical compound from an initial mixture to obtainthe compound in greater purity or at a higher concentration than thepurity or concentration of the compound in the initial mixture.

The term “aqueous phase,” as used herein, refers to the aqueous phase ofa biphasic mixture obtained by contacting a fermentation broth with awater-immiscible organic extractant. In an embodiment of a processdescribed herein that includes fermentative extraction, the term“fermentation broth” then specifically refers to the aqueous phase inbiphasic fermentative extraction.

The term “organic phase,” as used herein, refers to the non-aqueousphase of a biphasic mixture obtained by contacting a fermentation brothwith a water-immiscible organic extractant.

The terms “PDC-,” “PDC knockout,” or “PDC-KO” as used herein refer to acell that has a genetic modification to inactivate or reduce expressionof a gene encoding pyruvate decarboxylase (PDC) so that the cellsubstantially or completely lacks pyruvate decarboxylase enzymeactivity. If the cell has more than one expressed (active) PDC gene,then each of the active PDC genes can be inactivated or have minimalexpression thereby producing a PDC-cell.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to a nucleic acidmolecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).A polynucleotide can contain the nucleotide sequence of the full-lengthcDNA sequence, or a fragment thereof, including the untranslated 5′ and3′ sequences and the coding sequences. The polynucleotide can becomposed of any polyribonucleotide or polydeoxyribonucleotide, which canbe unmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatcan be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. “Polynucleotide” embraceschemically, enzymatically, or metabolically modified forms.

A polynucleotide sequence can be referred to as “isolated,” in which ithas been removed from its native environment. For example, aheterologous polynucleotide encoding a polypeptide or polypeptidefragment having dihydroxy-acid dehydratase activity contained in avector is considered isolated for the purposes of the present invention.Further examples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. An isolatedpolynucleotide fragment in the form of a polymer of DNA can be comprisedof one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “NAD(P)H consumption assay” refers to an enzyme assay for thedetermination of the specific activity of the KARI enzyme, involvingmeasuring the disappearance of the KARI cofactor, NAD(P)H, from theenzyme reaction. Such assays are described in Aulabaugh and Schloss,Biochemistry 29: 2824-2830, 1990, which is herein incorporated byreference in its entirety.

The term “NAD(P)H” refers to either NADH or NADPH.

“KARI” is the abbreviation for the enzyme ketol-acid reductoisomerase.

The term “close proximity” when referring to the position of variousamino acid residues of a KARI enzyme with respect to the adenosyl2′-phosphate of NADPH means amino acids in the three-dimensional modelfor the structure of the enzyme that are within about 4.5 Å of thephosphorus atom of the adenosyl 2′-phosphate at NADPH bound to theenzyme.

The term “ketol-acid reductoisomerase” (abbreviated “KARI”), and“acetohydroxy acid isomeroreductase” will be used interchangeably andrefer to enzymes capable of catalyzing the reaction of (S)-acetolactateto 2,3-dihydroxyisovalerate, classified as EC number EC 1.1.1.86 (EnzymeNomenclature 1992, Academic Press, San Diego). KARI is found in avariety of organisms and amino acid sequence comparisons across specieshave revealed that there are 2 types of this enzyme: a short form (classI) found in fungi and most bacteria, and a long form (class II) typicalof plants. Class I KARIs typically have between 330-340 amino acidresidues. The long form KARI enzymes have about 490 amino acid residues.However, some bacteria such as Escherichia coli possess a long form,where the amino acid sequence differs appreciably from that found inplants. KARI is encoded by the ilvC gene and is an essential enzyme forgrowth of E. coli and other bacteria in a minimal medium. Class II KARIsgenerally consist of a 225-residue N-terminal domain and a 287-residueC-terminal domain. The N-terminal domain, which contains thecofactor-binding site, has an αβstructure and resembles domains found inother pyridine nucleotide-dependent oxidoreductases. The C-terminaldomain consists almost entirely of α-helices.

KARI enzymes are described for example, in U.S. Pat. Nos. 7,910,342 and8,129,162 and U.S. Pub, App, No. 2010/0197519, all of which are hereinincorporated by reference in their entireties.

Ketol-acid reductoisomerase (KARI) enzymes are useful in pathways forthe production of isobutanol using engineered microorganisms (U.S. Pat.Nos. 7,851,188 and 7,993,889, incorporated by reference in theirentireties herein).

A KARI that can utilize NADH can capitalize on the NADH produced by theexisting glycolytic and other metabolic pathways in most commonly usedmicrobial cells and can result in improved isobutanol production. Raneet al. (Arch. Biochem. Biophys., 338: 83-89, 1997) discusses cofactorswitching of a ketol acid reductoisomerase isolated from E. coli. USAppl. Pub. Nos. 2009/0163376 and 2010/0197519 (each of which is hereinincorporated by reference it its entirety) describe variants of KARIenzymes which can use NADH. US Appl. Pub. No. 201010143997 (which isherein incorporated by reference in its entirety) describes E. colivariants with improved K_(M) values for NADH.

The terms “ketol-acid reductoisomerase activity” and “KARI activity”refer to the ability to catalyze the substrate to product conversion(S)-acetolactate to 2,3-dihydroxyisovalerate.

The term “acetolactate synthase” refers to an enzyme that is catalyzesthe conversion of pyruvate to acetolactate and CO₂.

Acetolactate has two stereoisomers ((R) and (S)); the enzyme prefers the(S)-isomer, which is made by biological systems. Certain acetolactatesynthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992,Academic Press, San Diego). These enzymes are available from a number ofsources, including, but not limited to, Bacillus subtilis (SEQ ID NO:165), Klebsiella pneumoniae (SEQ ID NO: 166) and Lactococcus lactis.

The term “acetohydroxy acid dehydratase” refers to an enzyme thatcatalyzes the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate. Certain acetohydroxy acid dehydratases are known bythe EC number 4.2.1.9. These enzymes are available from a vast array ofmicroorganisms, including, but not limited to, E. coli, S. cerevisiae,M. maripaludis, B. subtilis, Lactococcus lactis (SEQ ID NO:167), andStreptococcus mutans (SEQ ID NO: 168).

The term “branched-chain α-keto acid decarboxylase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyraldehydeand CO₂. Certain branched-chain α-keto acid decarboxylases are known bythe EC number 4.1.1.72 and are available from a number of sources,including, but not limited to, Lactococcus lactis (SEQ ID NO: 169),Salmonella typhimurium, Clostridium acetobutylicum, Macrococcuscaseolyticus (SEQ ID NO: 171), and Listeria grayi (SEQ ID NO: 170).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyraldehyde to isobutanol. Certainbranched-chain alcohol dehydrogenases are known by the EC number1.1.1.265, but can also be classified under other alcohol dehydrogenases(specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH(reduced nicotinamide adenine dinucleotide) and/or NADPH as electrondonor and are available from a number of sources, including, but notlimited to, S. cerevisiae, E. coli, C. acetobutylicum, B. indica (SEQ IDNO: 173), A. xylosoxidans (SEQ ID NO: 172).

As used herein, “DHMB” refers to 2,3-dihydroxy-2-methyl butyrate. DHMBincludes “fast DHMB,” which has the 2S, 3S configuration, and “slowDHMB,” which has the 2S, 3R configurate. See Kaneko et al.,Phytochemistry 39: 115-120 (1995), which is herein incorporated byreference in its entirety and refers to fast DHMB as angliceric acid andslow DHMB as tigliceric acid.

As used herein, “reduced activity” refers to any measurable decrease ina known biological activity of a polypeptide when compared to the samebiological activity of the polypeptide prior to the change resulting inthe reduced activity. Such a change can include a modification of apolypeptide or a polynucleotide encoding a polypeptide as describedherein. A reduced activity of a polypeptide disclosed herein can bedetermined by methods well known in the art and/or disclosed herein.

As used herein, “eliminated activity” refers to the complete abolishmentof a known biological activity of a polypeptide when compared to thesame biological activity of the polypeptide prior to the changeresulting in the eliminated activity. Such a change can include amodification of a polypeptide or a polynucleotide encoding a polypeptideas described herein. An eliminated activity includes a biologicalactivity of a polypeptide that is not measurable when compared to thesame biological activity of the polypeptide prior to the changeresulting in the eliminated activity. An eliminated activity of apolypeptide disclosed herein can be determined by methods well known inthe art and/or disclosed herein.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof. Non-limiting examples ofcarbon substrates are provided herein and include, but are not limitedto, monosaccharides, oligosaccharides, polysaccharides, ethanol,lactate, succinate, glycerol, carbon dioxide, methanol, glucose,fructose, sucrose, xylose, arabinose, dextrose, or mixtures thereof.Other carbon substrates can include ethanol, lactate, succinate, orglycerol.

“Fermentation broth” as used herein means the mixture of water, sugars(fermentable carbon sources), dissolved solids (if present),microorganisms producing alcohol, product alcohol and all otherconstituents of the material held in the fermentation vessel in whichproduct alcohol is being made by the reaction of sugars to alcohol,water and carbon dioxide (CO₂) by the microorganisms present. From timeto time, as used herein the term “fermentation medium” and “fermentedmixture” can be used synonymously with “fermentation broth”.

“Biomass” as used herein refers to a natural product containing ahydrolysable starch that provides a fermentable sugar, including anycellulosic or lignocellulosic material and materials comprisingcellulose, and optionally further comprising hemicellulose, lignin,starch, oligosaccharides, disaccharides, and/or monosaccharides. Biomasscan also comprise additional components, such as protein and/or lipids.Biomass can be derived from a single source, or biomass can comprise amixture derived from more than one source. For example, biomass cancomprise a mixture of corn cobs and corn stover, or a mixture of grassand leaves. Biomass includes, but is not limited to, bioenergy crops,agricultural residues, municipal solid waste, industrial solid waste,sludge from paper manufacture, yard waste, wood, and forestry waste.Examples of biomass include, but are not limited to, corn grain, corncobs, crop residues such as corn husks, corn stover, grasses, wheat,rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass,waste paper, sugar cane bagasse, sorghum, soy, components obtained frommilling of grains, trees, branches, roots, leaves, wood chips, sawdust,shrubs and bushes, vegetables, fruits, flowers, animal manure, andmixtures thereof.

“Feedstock” as used herein means a product containing a fermentablecarbon source. Suitable feedstock include, but are not limited to, rye,wheat, corn, sugar cane, and mixtures thereof.

The term “aerobic conditions” as used herein means growth conditions inthe presence of oxygen. Typically, oxygen is replaced by agitation ofthe medium such that gas exchange with the atmosphere occurs.

The term “microaerobic conditions” as used herein means growthconditions with low levels of oxygen (i.e., below normal atmosphericoxygen levels). “Microaerobic conditions” include conditions wherein themedium is not necessarily oxygen free when innoculated with amicroorganism, but, the oxygen present in the medium is consumed and itsreplacement by gas exchange is limited, for example, by limitingagitation and/or sealing the vessel from the atmosphere.

The term “anaerobic conditions” as used herein means growth conditionsin the absence of oxygen.

The term “isolated nucleic acid molecule”, “isolated nucleic acidfragment” and “genetic construct” will be used interchangeably and willmean a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAcan be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene can comprise regulatory sequences andcoding sequences to that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature.“Endogenous gene” refers to a native gene in its natural location in thegenome of a microorganism. A “foreign” gene refers to a gene notnormally found in the host microorganism, but that is introduced intothe host microorganism by gene transfer. Foreign genes can comprisenative genes inserted into a non-native microorganism, or chimericgenes. A “transgene” is a gene that has been introduced into the genomeby a transformation procedure.

As used herein, “native” refers to the form of a polynucleotide, gene,or polypeptide as found in nature with its own regulatory sequences, ifpresent.

As used herein the term “coding sequence” or “coding region” refers to aDNA sequence that encodes for a specific amino acid in sequence.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences can include promoters, is translationleader sequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites and stem-loop structures.

As used herein, “endogenous” refers to the native form of apolynucleotide, gene or polypeptide in its natural location in theorganism or in the genome of an organism. “Endogenous polynucleotide”includes a native polynucleotide in its natural location in the genomeof an organism. “Endogenous gene” includes a native gene in its naturallocation in the genome of an organism. “Endogenous polypeptide” includesa native polypeptide in its natural location in the organism transcribedand translated from a native polynucleotide or gene in its naturallocation in the genome of an organism.

The term “heterologous” when used in reference to a polynucleotide, agene, or a polypeptide refers to a polynucleotide, gene, or polypeptidenot normally found in the host organism. “Heterologous” also includes anative coding region, or portion thereof, that is reintroduced into thesource organism in a form that is different from the correspondingnative gene, e.g., not in its natural location in the organism's genome.The heterologous polynucleotide or gene can be introduced into the hostorganism by, e.g., gene transfer. A heterologous gene can include anative coding region with non-native regulatory regions that isreintroduced into the native host. For example, a heterologous gene caninclude a native coding region that is a portion of a chimeric geneincluding non-native regulatory regions that is reintroduced into thenative host. “Heterologous polypeptide” includes a native polypeptidethat is reintroduced into the source organism in a form that isdifferent from the corresponding native polypeptide.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure.

As used herein, the term “modification” refers to a change in apolynucleotide disclosed herein that results in reduced or eliminatedactivity of a polypeptide encoded by the polynucleotide, as well as achange in a polypeptide disclosed herein that results in reduced oreliminated activity of the polypeptide. Such changes can be made bymethods well known in the art, including, but not limited to, deleting,mutating (e.g., spontaneous mutagenesis, random mutagenesis, mutagenesiscaused by mutator genes, or transposon mutagenesis), substituting,inserting, down-regulating, altering the cellular location, altering thestate of the polynucleotide or polypeptide (e.g., methylation,phosphorylation or ubiquitination), removing a cofactor, introduction ofan antisense RNA/DNA, introduction of an interfering RNA/DNA, chemicalmodification, covalent modification, irradiation with UV or X-rays,homologous recombination, mitotic recombination, promoter replacementmethods, and/or combinations thereof. Guidance in determining whichnucleotides or amino acid residues can be modified, can be found bycomparing the sequence of the particular polynucleotide or polypeptidewith that of homologous polynucleotides or polypeptides, e.g., yeast orbacterial, and maximizing the number of modifications made in regions ofhigh homology (conserved regions) or consensus sequences.

“Regulatory sequences” refers to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences can include promoters, enhancers,operators, repressors, transcription termination signals, translationleader sequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure.

The term “promoter” refers to a nucleic acid sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence,Promoters can be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic nucleic acid segments. It isunderstood by those skilled in the art that different promoters candirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental or physiological conditions. Promoters which cause a geneto be expressed in most cell types at most times are commonly referredto as “constitutive promoters”. “Inducible promoters,” on the otherhand, cause a gene to be expressed when the promoter is induced orturned on by a promoter-specific signal or molecule. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths can have identical promoter activity. For example, it will beunderstood that “FBA1 promoter” can be used to refer to a fragmentderived from the promoter region of the FBA1 gene.

The term “terminator” as used herein refers to DNA sequences locateddownstream of a coding sequence. This includes polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The 3′ region can influence the transcription, RNA processingor stability, or translation of the associated coding sequence. It isrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths can have identical terminator activity. For example, it will beunderstood that “CYC1 terminator” can be used to refer to a fragmentderived from the terminator region of the CYC1 gene.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression can also refer totranslation of mRNA into a polypeptide.

The term “overexpression,” as used herein, refers to expression that ishigher than endogenous expression of the same or related gene. Aheterologous gene is overexpressed if its expression is higher than thatof a comparable endogenous gene. The term overexpression refers to anincrease in the level of nucleic acid or protein in a host cell. Thus,overexpression can result from increasing the level of transcription ortranslation of an endogenous sequence in a host cell or can result fromthe introduction of a heterologous sequence into a host cell.Overexpression can also result from increasing the stability of anucleic acid or protein sequence.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into the genome of a host microorganism, resultingin genetically stable inheritance. Host microorganisms containing thetransformed nucleic acid fragments are referred to as “transgenic” or“recombinant” or “transformed” microorganisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements can be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derive from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withoutaffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S)TAT Tyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGCTTA Leu (L) TCA Ser (S) TAA Stop TGA Stop TTG Leu (L) TCG Ser (S)TAG Stop TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R)CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P)CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R)A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I)ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K)AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R) GGTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala (A)GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G)GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code m forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available atwww.kazusa.or.jp/codon/ (visited Mar. 20, 2008), and these tables can beadapted in a number of ways. See Nakamura, Y., et al, Nucl. Acids Res.28:292 (2000). Codon usage tables for yeast, calculated from GenBankRelease 128.0 [15 Feb. 2002], are reproduced below as Table 2B. Thistable uses mRNA nomenclature, and so instead of thymine (T) which isfound in DNA, the tables use uracil (U) which is found in RNA. Table 2has been adapted so that frequencies are calculated for each amino acid,rather than for all 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Frequency perAmino Acid Codon Number thousand Phe UUU 170666 26.1 Phe UUC 120510 18.4Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC 355455.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 Ile AUC112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 144243 22.1Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU 15355723.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 Ser AGU92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8 ProCCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 83207 12.7Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC 8235712.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 Tyr UAC96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3 GlnCAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA 27361841.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2 Glu GAA297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 31095 4.8 TrpUGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 19562 3.0 ArgCGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU 156109 23.9Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 Stop UAA 6913 1.1Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence, can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs are readily available to those of ordinaryskill in the art. For example, the “EditSeq” function in the LasergenePackage, available from DNAstar, Inc., Madison, Wis., thebacktranslation function in the VectorNTI Suite, available fromInforMax, Inc., Bethesda, Md., and the “backtranslate” function in theGCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif.In addition, various resources are publicly available to codon-optimizecoding region sequences, e.g., the “backtranslation” function atwww.entelechon.com/bioinformatics/backtranslation.php?lang=eng (visitedApr. 15, 2008) and the “backtranseq” function available athttp://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 9, 2002).Constructing a rudimentary algorithm to assign codons based on a givenfrequency can also easily be accomplished with basic mathematicalfunctions by one of ordinary skill in the art.

Codon-optimized coding regions can be designed by various methods knownto those skilled in the art including software packages such as“synthetic gene designer” (userpages.umbc.edu/˜wug1/codon/sgd/, visitedMar. 19, 2012).

A polynucleotide or nucleic acid fragment is “hybridizable” to anothernucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule,when a single-stranded form of the nucleic acid fragment can anneal tothe other nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post hybridization washes determinestringency conditions. One set of conditions uses a series of washesstarting with 6×SSC, 0.5% SDS at room temperature for 15 min, thenrepeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeatedtwice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. Another set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C. An additional set of stringent conditions includehybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1%SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50 9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7 11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. In one embodiment, a minimum length for a hybridizablenucleic acid is at least about 15 nucleotides; at least about 20nucleotides; or the length is at least about 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration can be adjusted as necessary accordingto factors such as length of the probe.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein”, “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. Apolypeptide can be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis. Suitable methods for polypeptidesynthesis are known in the art.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, the terms “variant” and “mutant” are synonymous andrefer to a polypeptide differing from a specifically recited polypeptideby one or more amino acid insertions, deletions, mutations, andsubstitutions, created using, e.g., recombinant DNA techniques, such asmutagenesis. Guidance in determining which amino acid residues can bereplaced, added, or deleted without abolishing activities of interest,can be found by comparing the sequence of the particular polypeptidewith that of homologous polypeptides, e.g., yeast or bacterial, andminimizing the number of amino acid sequence changes made in regions ofhigh homology (conserved regions) or by replacing amino acids withconsensus sequences.

“Engineered polypeptide” as used herein refers to a polypeptide that issynthetic, i.e., differing in some manner from a polypeptide found innature.

Alternatively, recombinant polynucleotide variants encoding these sameor similar polypeptides can be synthesized or selected by making use ofthe “redundancy” in the genetic code. Various codon substitutions, suchas silent changes which produce various restriction sites, can beintroduced to optimize cloning into a plasmid or viral vector forexpression. Mutations in the polynucleotide sequence can be reflected inthe polypeptide or domains of other peptides added to the polypeptide tomodify the properties of any part of the polypeptide. For example,mutations can be used to reduce or eliminate expression of a targetprotein and include, but are not limited to, deletion of the entire geneor a portion of the gene, inserting a DNA fragment into the gene (ineither the promoter or coding region) so that the protein is notexpressed or expressed at lower levels, introducing a mutation into thecoding region which adds a stop codon or frame shift such that afunctional protein is not expressed, and introducing one or moremutations into the coding region to alter amino acids so that anon-functional or a less enzymatically active protein is expressed.

Amino acid “substitutions” can be the result of replacing one amino acidwith another amino acid having similar structural and/or chemicalproperties, i.e., conservative amino acid replacements, or they can bethe result of replacing one amino acid with an amino acid havingdifferent structural and/or chemical properties, i.e., non-conservativeamino acid replacements. “Conservative” amino acid substitutions can bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid. Alternatively, “non-conservative” amino acidsubstitutions can be made by selecting the differences in polarity,charge, solubility, hydrophobicity, hydrophilicity, or the amphipathicnature of any of these amino acids. “Insertions” or “deletions” can bewithin the range of variation as structurally or functionally toleratedby the recombinant proteins. The variation allowed can be experimentallydetermined by systematically making insertions, deletions, orsubstitutions of amino acids in a polypeptide molecule using recombinantDNA techniques and assaying the resulting recombinant variants foractivity.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul, S. F., et al.,J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten ormore contiguous amino acids or thirty or more nucleotides is necessaryin order to putatively identify a polypeptide or nucleic acid sequenceas homologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides can be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases can be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches the complete amino acidand nucleotide sequence encoding particular proteins. The skilledartisan, having the benefit of the sequences as reported herein, can nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenine is complementary to thymine andcytosine is complementary to guanine, and with respect to RNA, adenineis complementary to uracil and cytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Methods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations can be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences are performed using the “Clustal method of alignment” whichencompasses several varieties of the algorithm including the “Clustal Vmethod of alignment” corresponding to the alignment method labeledClustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989);Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) andfound in the MegAlignn™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc.). For multiple alignments, the defaultvalues correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After alignment of the sequences using the Ciustal V program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” table in the same program. Additionally the “Ciustal W methodof alignment” is available and corresponds to the alignment methodlabeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992))and found in the MegAlign™ v6.1 program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc.). Default parameters for multiplealignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay DivergenSeqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=GonnetSeries, DNA Weight Matrix=IUB). After alignment of the sequences usingthe Clustal W program, it is possible to obtain a “percent identity” byviewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides wherein suchpolypeptides have the same or similar function or activity, or indescribing the corresponding polynucleotides. Useful examples of percentidentities include, but are not limited to: 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% can beuseful in describing the present invention, such as 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.Suitable polynucleotide fragments not only have the above homologies buttypically comprise a polynucleotide having at least 50 nucleotides, atleast 100 nucleotides, at least 150 nucleotides, at least 200nucleotides, or at least 250 nucleotides. Further, suitablepolynucleotide fragments having the above homologies encode apolypeptide having at least 50 amino acids, at least 100 amino acids, atleast 150 amino acids, to at least 200 amino acids, or at least 250amino acids. Polynucleotides can be cloned or synthesized using methodsknown in the art.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” can be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques are well knownin the art and are described by Sambrook, J., Fritsch, E. F. andManiatis, T., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. andEnquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987). Additional methods usedhere are in Methods in Enzymology, Volume 194, Guide to Yeast Geneticsand Molecular and Cell Biology (Part A, 2004, Christine Guthrie andGerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).Other molecular tools and techniques are known in the art and includesplicing by overlapping extension polymerase chain reaction (PCR) (Yu,et al. (2004) Fungal Genet. Biol. 41:973-981), positive selection formutations at the URA3 locus of Saccharomyces cerevisiae (Boeke, J. D. etal. (1984) Mol. Gen. Genet. 197, 345-346; M A Romanos, et al. NucleicAcids Res. 1991 January 11; 19(1): 187), the cre-lox site-specificrecombination system as well as mutant lox sites and FLP substratemutations (Sauer, B. (1987) Mol Cell Biol 7: 2087-2096; Senecoff, et al.(1988) Journal of Molecular Biology, Volume 201, Issue 2, Pages 405-421;Albert, et al. (1995) The Plant Journal. Volume 7, Issue 4, pages649-659), “seamless” gene deletion (Akada, et al. (2006) Yeast;23(5):399-405), and gap repair methodology (Ma et al., Genetics58:201-216; 1981).

The genetic manipulations of a recombinant host cell disclosed hereincan be performed using standard genetic techniques and screening and canbe made in any host cell that is suitable to genetic manipulation(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 201-202).

In embodiments, a recombinant host cell disclosed herein can be anyyeast or fungi host useful for genetic modification and recombinant geneexpression including those yeast mentioned elsewhere herein. In otherembodiments, a recombinant host cell can be a member of the generaIssatchenkia, Zygosaccharomyces, Saccharomyces, Schizosaccharomyces,Dekkera, Torulopsis, Brettanomyces, Torulaspora, Hanseniaspora,Kluveromyces, Yarrowia, and some species of Candida.

Polypeptides with KARI Activity Suited for Biosynthetic Pathways

US Appl. Pub. No. 2010/0197519A1 describes variants of the Pseudomonasfluorescens KARI enzyme suitable for isobutanol production, including avariant “JEA1” (SEQ ID NO: 2) which was demonstrated to have a lowerK_(M) for the cofactor NADH than the wild-type enzyme.

As demonstrated in the Examples herein, variants of JEA1 have beenprovided which are believed to be particularly suitable for use inrecombinant microbial host cells comprising an isobutanol biosyntheticpathway, including yeast cells such as S. cerevisiae and in methodsutilizing such host cells under anaerobic conditions. As such, thevariants provided herein may also be useful in other biosyntheticpathways comprising a substrate to product conversion catalyzed by KARIactivity.

Based on the results given in the Examples, substitutions at one ormore, two or more, three or more, four or more, five or more, six ormore, seven or more, or eight or more of the following positions of SEQID NO: 2 give rise to polypeptides suited for use in recombinant hostcells comprising an isobutanol biosynthetic pathway: Y286, I152, S322,S292, I291, I13, P47, F50, V53, A268, V76, A336, L88, A71, G72, M301,Y239, Y113, N114, A329, A69, A295, E264, R280, S157, M238, Q272, K335,K99, R275, R306.

In embodiments, substitutions at these positions include, but are notlimited to, those demonstrated in the Examples: Y286F, A336R, I152V,S322A, S292A, 1291V, I13L, P47Y, F50A, V53A, A268E, V76I, A336G, L88V,A71K, G72W, I152V, A268T, A329E, M301I, Y239H, Y113F, S322A, N114G,A329R, A69T, N114S, A295V, E264K, R280D, S157T, M2381, Q272T, K335M,R280H, K99N, R275K, R306K or a combination thereof.

Combinations of substitutions may include, but are not limited to, thosedemonstrated in Examples 1-5, Tables 9, 11, 13, 15, and 17. Asdemonstrated, certain of the combinations of substitutions are suitablefor use in recombinant microbial host cells comprising an isobutanolbiosynthetic pathway.

For example, as shown in Example 2, a suitable combination is Y286,A336, and 1152. Example variants with substitutions in these positionsare demonstrated in the Examples. In embodiments, the substitutions atthese positions are Y286F, A336R, and I152V. Another suitablecombination is A68, I152, Y286 and A336. In embodiments, thesubstitutions at these positions are A68E, I152V, Y286F, and A336R. Anexample variant containing those substitutions is given as SEQ ID NO:346 (“R8-SOG1_y2”).

Other combinations comprise substitutions at one or more of thefollowing sites: A69, A71, Y113, M238, Y239, M301, A322, or A329.Examples of such combinations are shown in Example 5.

In embodiments, for variants provided herein, including variants incomprising substitutions at I152, Y286 and A336 and optionally A68substitutions may be selected from those indicated with an “X” in Table3 for the indicated positions:

TABLE 3 Substitutions for KARI variants Amino Acid Position 152 Position286 Position 336 A, Ala X X X C, Cys X X X D, Asp X — X E, Glu X — X F,Phe X X X G, Gly X — X H, His X X X I, Ile X X X K, Lys X — X L, Leu X —X M, Met X X X N, Asn X — X P, Pro X — X Q, Gln X — X R, Arg X — X S,Ser X — X T, Thr X — X V, Val X — X W, Trp X X X Y, Tyr X X X

Equipped with this disclosure, one of skill in the art can readily makeand use additional substitutions at any of the indicated sites (Y286,I152, S322, S292, I291, I13, P47, F50, V53, A268, V76, A336, L88, A71,G72, M301, Y239, Y113, N114, A329, A69, A295, E264, R280, S157, M238,Q272, K335, K99, R275, and R306.) to produce additional variants.Methods of generating such variants are demonstrated herein (see Example6) and/or are known in the art as described below. In one embodiment,conservative substitutions are made based on the sequences exemplifiedherein. In another embodiment, all or a subset of amino acids aresubstituted at a given site and screened for activity.

In embodiments, in polypeptides provided herein: the amino acid at theposition corresponding to position 24 of SEQ ID NO: 2 is F; the aminoacid at the position corresponding to position 33 of SEQ ID NO: 2 is L;the amino acid at the position corresponding to position 47 of SEQ IDNO: 2 is P; the amino acid at the position corresponding to position 50of SEQ ID NO: 2 is F; the amino acid at the position corresponding toposition 61 of to SEQ ID NO: 2 is F; the amino acid at the positioncorresponding to position 80 of SEQ ID NO: 2 is I; and the amino acid atthe position corresponding to position 156 of SEQ ID NO: 2 is V.

It will be appreciated that using a combination of structural andsequence information available in the art, polypeptides comprising KARIactivity and less than 100% identity to the exemplified sequences can beconstructed for use in isobutanol or other biosynthetic pathways. Forexample, crystal structures of the E. coli KARI enzyme at 2.6 Åresolution have been solved (Tyagi, et al., Protein Sci., 14: 3089-3100,2005) as has the structure of the P. aeruginosa KARI (Ahn, et al., J.Mol. Biol., 328: 505-515, 2003) and the KARI enzyme from spinach (BiouV., et al. The EMBO Journal, 16: 3405-3415, 1997). Furthermore, a KARIProfile HMM prepared using amino acid sequences of 25 KARI proteins withexperimentally verified function is described in U.S. Pat. No.8,129,162. The KARIs were from Pseudomonas fluorescens Pf-5, Sulfolobussolfataricus P2, Pyrobaculum aerophilum str. 1M2, Natronomonas pharaonisDSM 2160, Bacillus subtilis subsp. subtilis str. 168, Corynebacteriumglutamicum ATCC 13032, Phaeospririlum molischianurn, Ralstoniasolanacearum GMI1000, Zymomonas mobilis subsp. mobilis ZM4,Alkalilimnicola ehrhichei MLHE-, Campylobacter lari RM2100, Marinobacteraquaeolei VT8, Psychrobacter arcticus 273-4, Hahella chejuensis KCTC2396, Thiobacillus denitrificans ATCC 25259, Azotobacter vinelandiiAvOP, Pseudomonas syringae pv. syringae B728a, Pseudomonas syringae pv.tomato str. DC3000, Pseudomonas putida KT2440, Pseudomonas entomophilaL48, Pseudomonas mendocina ymp, Pseudomonas aeruginosa PAO1, Bacilluscereus ATCC 10987, Bacillus cereus ATCC 10987, and Spinacia oleracea.Any protein that matches the KARI Profile HMM (described in U.S. Pat.No. 8,129,162 and incorporated by reference in its entirety herein) withan E value of <10⁻³ using hmmsearch program in the HMMER package isexpected to be a functional KARI.

As shown in FIGS. 9-12, substitution positions disclosed herein can begrouped according to their structural location using homology modelingof PF5 KARI as disclosed in U.S. Pat. No. 8,129,162. As disclosedtherein, the structure of PF5-KARI with bound NADPH, acetolactate andmagnesium ions was built based on the crystal structure of P. aeruginosaPAO1-KARI (PDB ID 1NP3, Ahn H. J. et al, J. Mol. Biol., 328, 505-515,2003) which has 92% amino acid sequence identity to PF5 KARI. PAO1-KARIstructure is a homo-dodecamer and each dodecamer consists of sixhomo-dimers with extensive dimer interface. The active site of KARI islocated in this dimer interface. The biological assembly is formed bysix homo-dimers positioned on the edges of a tetrahedron resulting in ahighly symmetrical dodecamer of 23 point group symmetry. For simplicity,only the dimeric unit (monomer A and monomer B) was built for thehomology model of PF5-KARI because the active site is in the homo-dimerinterface.

The model of PF5-KARI dimer was built based on the coordinates ofmonomer A and monomer B of PAO1-KARI and sequence of PF5-KARI usingDeepView/Swiss PDB viewer (Guex, N. and Peitsch, M. C. Electrophoresis18: 2714-2723, 1997). This model was then imported to program O (Jones,T. A. et al, Acta Crystallogr. A 47, 110-119, 1991) on a SiliconGraphics system for further modification.

The structure of PAO1-KARI has no cofactor, substrate or inhibitor ormagnesium in the active site. Therefore, the spinach KARI structure (PDBID 1yve, Biou V. et al. The EMBO Journal, 16: 3405-3415, 1997.), whichhas magnesium ions, NADPH and inhibitor (N-Hydroxy-N-isopropyloxamate)in the acetolactate binding site, was used to model these molecules inthe active site. The plant KARI has very little sequence identity toeither PF5- or PAO1 KARI (<20% amino acid identity), however thestructures in the active site region of these two KARI enzymes are verysimilar. To overlay the active site of these two KARI structures,commands LSQ_ext, LSQ_improve, LSQ_mol in the program O were used toline up the active site of monomer A of spinach KARI to the monomer A ofPF5 KARI model. The coordinates of NADPH, two magnesium ions and theinhibitor bound in the active site of spinach KARI were extracted andincorporated to molecule A of PF5 KARL A set of the coordinates of thesemolecules was generated for monomer B of PF5 KARI by applying thetransformation operator from monomer A to monomer B calculated by theprogram.

Because there is no cofactor in the active site of PAO1 KARI crystalstructure, the structures of the phosphate binding loop region in thecofactor binding site (residues 44-45 in PAO1 KARI, 157-170 in spinachKARI) are very different between the two. To model the cofactor-boundform, the model of the PF5-KARI phosphate binding loop (44-55) wasreplaced by that of 1yve (157-170). Any discrepancy of side chainsbetween these two was converted to those in the PF5-KARI sequence usingthe mutate_replace command in program O, and the conformations of thereplaced side-chains were manually adjusted. The entireNADPH/Mg/inhibitor bound dimeric PF5-KARI model went through one roundof energy minimization using program CNX (ACCELRYS San Diego Calif.,Burnger, A. T. and Warren, G. L., Acta Crystallogr., D 54, 905-921,1998) after which the inhibitor was replaced by the substrate,acetolactate (AL), in the model. The conformation of AL was manuallyadjusted to favor hydride transfer between C4 of the nicotinamide ofNADPH and the substrate. No further energy minimization was performed onthis model.

The C-terminal tail (328-338) is not seen in the model and is disorderedin the crystal structure PDB code 1np3 that was used to build thehomology model of Pf5-KARI. The C-terminal tail has been shown to beimportant for the binding of cofactor and substrate during catalysis(JMB (2009)389, 167-182 Leung & Guddat).The following positions are inthe C-terminal tail region: A329, K335, A336.

As shown in FIG. 10, positions M238, Y239, E264, N267, A268, Q272, A277,R280, Y286, I291, S292, M301, S322, are associated with theinter-molecular dimer interface region, and positions Y113, N114, I291,S292, and A295 are associated with the inter-domain interface region.

Positions of P47, P50, V53, T80, and L88 are part of the modelednucleotide binding region (FIG. 11A), Positions 68, 69, 71 and 72 areassociated with N-domain surface helix (positions 64-73; FIG. 11B).

Positions C33, L61, I127, I152, and V171 are in the N-domain hydrophobicbridge region, shown in FIG. 12.

Accordingly, provided herein are variants comprising substitutions in atleast one of the inter-molecular dimer interface region, inter-domaininterface region, nucleotide binding region, N-domain surface helixregion, hydrophobic bridge region, and the C-terminal tail region. Inembodiments, variants provided herein comprise substitutions in thenucleotide binding region and substitutions in at least one of, at leasttwo of, or at least three of the following regions: the inter-moleculardimer interface region, inter-domain interface region, N-domain surfacehelix region, hydrophobic bridge region, or the C-terminal tail region.

The sequences of other polynucleotides, genes and/or polypeptides can beidentified in the literature and in bioirformatics databases well knownto the skilled person using sequences disclosed herein and available inthe art. For example, such sequences can be identified through BLASTsearching of publicly available databases with polynucleotide orpolypeptide sequences provided herein. In such a method, identities canbe based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix.

Additionally, polynucleotide or polypeptide sequences disclosed hereincan be used to identify other KARI homologs in nature. For example, eachof the KARI encoding nucleic acid fragments disclosed herein can be usedto isolate genes encoding homologous proteins. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limited to(1) methods of nucleic acid hybridization; (2) methods of DNA and RNAamplification, as exemplified by various uses of nucleic acidamplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and (3) methods of library construction andscreening by complementation.

It will be appreciated that one of ordinary skill in the art, equippedwith this disclosure as well as sequence and structural informationavailable herein and/or in the art, can also generate active fragmentsof to polypeptides provided herein, for example, by truncatingpolypeptides provided herein at the N-terminus or C-terminus andconfirming KARI activity.

Accordingly, provided herein are polypeptides having at least about 90%identity to, at least about 95% identity to, at least about 99% identityto, or having the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 116,118, 119, 120, 121, 123, 124, 128, 129, 131, 132, 133, 134, 136, 137,138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 157, 159, 160, 161, or 346 or an active fragment thereof.

Generation of Variants

Variants described herein may be generated by any method known in theart. Methods known in the art for site-directed mutagenesis include, forexample, QuikChange® (Agilent, Santa Clara, Calif.) and Change-IT®(Affymetrix/USB, Santa Clara, Calif.). Methods known in the art forrandom point mutagenesis include, for example, error-prone PCR (e.g.,Bloom et al., BMC Biol. 2007, 5:29, doi:10.1186/1741-7007-5-29.) orGeneMorph®(Agilent, Santa Clara, Calif.), exposure to chemical mutagens(e.g., ethyl methanesulfonate) or ultraviolet light, use of modifiednucleotides in PCR (e.g., Wong et al., Nucleic Acids Res. 2004, 32:3,e26.), and use of special mutator strains. Methods known in the art forDNA recombination or “shuffling” include, for example, randomfragmentation and reassembly (e.g. Stemmer 1994 Proc. Natl. Acad. Sci.USA 91:22, 10747-10751.), heteroduplex repair (e.g., Volkov et al.,Nucleic Acids Res. 1999 27:18, e18.), staggered extension (e.g., Zhao etal., Nat. Biotechnol. 1998, 16:3, 258-261.), unpaired-primer shuffling(e.g., Milano et al., U.S. Pat. No. 7,879,582), site-directedrecombination (e.g., Hiraga et al., J. Mol. Biol. 2003, 330:2,287-296.), and synthetic shuffling (e.g., Ness et al., Nat. Biotechnol.2002, 20, 1251-1255.). Other methods for protein variant libraryconstruction include, for example, circular permutation (e.g., Guntas etal., PLoS One, 2012, 7(4):e35998), and chemical DNA synthesis.

Equipped with this disclosure, one of skill in the art can readily makeand use the variants provided herein as well as variants with less than100% identity (as described above) thereto.

KARI Activity

Polypeptides described herein include those with KARI activity. KARIactivity can be confirmed by assaying for the enzymatic conversion ofacetolactate to 2,3-dihydroxyisovalerate using methods described in theart (for example in U.S. Pat. No. 8,129,162, incorporated herein byreference). For example, the conversion may be followed by measuring thedisappearance of the cofactor, NADPH or NADH, from the reaction at 340nm using a plate reader (such as from Molecular Device, Sunnyvale,Calif.).

KARI activity may also be confirmed by expressing a given KARI in a hostcell comprising polynucleotides encoding polypeptides that catalyze thesubstrate to product conversions given in FIG. 1, steps a, c, d, and eand measuring the production of isobutanol, as described anddemonstrated herein (see Examples). Alternatively, KARI activity may beconfirmed by measuring the production of intermediate products in thebiosynthetic pathway downstream of the substrate to product conversioncatalyzed by KARI. Likewise, host cells comprising the substrate toproduct conversions for other biosynthetic pathways can also be used toconfirm KARI activity using a like strategy and confirming theproduction of the biosynthetic pathway product or intermediate productsdownstream of the substrate to product conversion catalyzed by KARI.

Confirmation of Isobutanol Production

The presence and/or concentration of isobutanol in the culture mediumcan be determined by a number of methods known in the art (see, forexample, U.S. Pat. No. 7,851,188, incorporated by reference in itsentirety). For example, a specific high performance liquidchromatography (HPLC) method utilizes a Shodex SH-1011 column with aShodex SHG guard column, both may be purchased from Waters Corporation(Milford, Mass.), with refractive index (RI) detection. Chromatographicseparation is achieved using 0.01 M H₂SO₄ as the mobile phase with aflow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanolhas a retention time of 46.6 min under the conditions used.

Alternatively, gas chromatography (GC) methods are available. Forexample, a specific GC method utilizes an HP-INNOWax column (30 m×0.53mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.),with a flame ionization detector (FID). The carrier gas is helium at aflow rate of 4.5 mL/min, measured at 150° C. with constant headpressure; injector split is 1:25 at 200° C.; oven temperature is 45° C.for 1 min, 45 to 220° C. at 10° C./min, and 220° C. for 5 min; and FIDdetection is employed at 240° C. with 26 mL/min helium makeup gas. Theretention time of isobutanol is 4.5 min.

Reduction of DHMB

The production of DHMB in a host cell comprising an isobutanolbiosynthetic pathway indicates that not all of the pathway substratesare being converted to the desired product. Thus, yield is lowered. Inaddition, DHMB can have inhibitory effects on product production. Forexample, DHMB can decrease the activity of enzymes in the biosyntheticpathway or have other inhibitory effects on yeast growth and/orproductivity during fermentation. Thus, the methods described hereinprovide ways of reducing DHMB during fermentation. The methods includeboth methods of decreasing the production of DHMB and methods ofremoving DHMB from fermenting compositions.

Decreasing DHMB Production

In some embodiments described herein, a recombinant host cell cancomprise reduced or eliminated ability to convert acetolactate to DHMB.The ability of a host cell to convert acetolactate to DHMB can bereduced or eliminated, for example, by a modification or disruption of apolynucleotide or gene encoding a polypeptide having acetolactatereductase activity or a modification or disruption of a polypeptidehaving acetolactate reductase activity. In other embodiments, therecombinant host cell can comprise a deletion, mutation, and/orsubstitution in an endogenous polynucleotide or gene encoding apolypeptide having acetolactate reductase activity or in an endogenouspolypeptide having acetolactate reductase. Such modifications,disruptions, deletions, mutations, and/or substitutions can result inacetolactate reductase activity that is reduced, substantiallyeliminated, or eliminated. In some embodiments of the invention, theproduct of the biosynthetic pathway is produced at a greater yield oramount compared to the production of the same product in a recombinanthost cell that does not comprise reduced or eliminated ability toconvert acetolactate to DHMB.

Thus, the product can be a composition comprising isobutanol that issubstantially free of, or free of DHMB. In some embodiments, thecomposition comprising butanol contains no more than about 5 mM, about 4mM, about 3 mM, about 2 mM, about 1 mM, about 0.5 mM, about 0.4 mM,about 0.3 mM DHMB, or about 0.2 mM DHMB.

Any product of a biosynthetic pathway that involves the conversion ofacetolactate to a substrate other than DHMB can be produced with greatereffectiveness in a recombinant host cell disclosed herein having thedescribed modification of acetolactate reductase activity. Such productsinclude, but are not limited to, butanol, e.g., isobutanol, 2-butanol,and BDO, and branched chain amino acids.

In some embodiments, the host cell comprises at least one deletion,mutation, and/or substitution in at least one endogenous polynucleotideencoding a polypeptide having acetolactate reductase activity. In someembodiments, the host cell comprises at least one deletion, mutation,and/or substitution in each of at least two endogenous polynucleotidesencoding polypeptides having acetolactate reductase activity.

In some embodiments, a polypeptide having acetolactate reductaseactivity can catalyze the conversion of acetolactate to DHMB. In someembodiments, a polypeptide having acetolactate reductase activity iscapable of catalyzing the reduction of acetolactate to 2S,3S-DHMB (fastDHMB) and/or 2S,3R-DHMB (slow DHMB).

TABLE 4 Polypeptides and polynucleotides having acetolactate reductaseactivity in Saccharomyces cerevisiae SEQ ID NO: (nucleic acid, Geneamino acid) YMR226C 174, 175 YIL074C (Chr 9) 176, 177 YIR036C (Chr 9)178, 179 YPL061W (ALD6)(Chr 16) 180, 181 YPL088W(Chr 16) 182, 183YCR105W (ADH7)(Chr 3) 184, 185 YDR541C(Chr 4) 186, 187 YER081 (SER3)(Chr5) 188, 189 YPL275W (FDH2)(Chr 16) 190, 191 YBR006W (UGA5)(Chr2) 192,193 YOL059W (Chr 15) 194, 195 YER081W (Chr 5) 196, 197 YOR375C (Chr 15)198, 199

In some embodiments, the conversion of acetolactate to DHMB in arecombinant host cell is reduced, substantially eliminated, oreliminated.

In some embodiments, the polypeptide having acetolactate reductaseactivity is selected from the group consisting of: YMR226C, YER081W,YIL074C, YBR006W, YPL275W, YOL059W, YIR036C, YPL061W, YPL088W, YCR105W,YOR375C, and YDR541C. In some embodiments, the polypeptide havingacetolactate reductase activity is a polypeptide comprising a sequencelisted in Table 4 or a sequence that is at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 99% identical to a polypeptidesequence listed in Table 4. In some embodiments, the polypeptide havingacetolactate reducatase activity is a polypeptide encoded by apolynucleotide sequence listed in Table 4 or a sequence that is at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 99% identicalto a polynucleotide sequence listed in Table 4.

TABLE 5 Example YMR226C Yeast Homologs SEQ ID NO: (nucleic SpeciesAccession # acid, amino acid) Saccharomyces paradoxus AABY01000127200, 201 Saccharomyces bayanus AACA01000631 202, 203MSQGRKAAERLANKTVLITGAS AGIGKATALEYLEASNGNMKLIL AARRLEKLEELKKTIDEEFPNAKVHVGQLDITQAEKIKPFIENLPEA FKDIDILINNAGKALGSERVGEIATQDIQDVFDTNVTALINVTQAVL PIFQAKNSGDIVNLGLGGRQRRIP HRLHLLCFQVCRRCVH*QFEKGTDQHEDQSYLDRAGAG*DRVLTG QIQR**GTS*KRLQGHYAVDGRR RG*LNRIFHFQKAEHRGCRHPDLPHQPSLALPHLSRL* (SEQ ID NO: 701) The sequence came from acomparative genomics study using “draft” genome sequences with 7-fold coverage (Kellis et al, Nature 423: 241-254 2003)).Saccharomyces castellii AACF01000116 204, 205 Saccharomyces mikataeAACH01000019 206, 207 Ashbya gossypii AE016819 208, 209 Candida glabrataCR380959 210, 211 Debaryomyces hansenii CR382139 212, 213Scheffersomyces stipitis XM_001387479 214, 215(formerly Pichia stipitis) Meyerozyma guilliermondii XM_001482184216, 217 (formerly Pichia guilliermondii) Vanderwaltozyma polysporaXM_001645671 218, 219 (formerly Kluyveromyces polysporus)Candida dubliniensis XM_002419771 220, 221 Zygosaccharomyces rouxiiXM_002494574 222, 223 Lachancea thermotolerans XM_002553230 224, 225(formerly Kluyveromyces thermotolerans) Kluyveromyces lactis XM_451902226, 227 Saccharomyces kluyveri SAKL0H04730 228, 229 Yarrowia lipolyticaXM_501554 230, 231 Schizosaccharomyces pombe NM_001018495 232, 233

In some embodiments, a polypeptide having acetolactate reductaseactivity is YMR226C or a homolog of YMR226C. Thus, in some embodiments,the polypeptide having acetolactate reductase activity is a polypeptidecomprising a sequence listed in Table 5 or a sequence that is at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 99% identicalto a polypeptide sequence listed in Table 5. In some embodiments, thepolypeptide having acetolactate reductase activity is a polypeptideencoded by a polynucleotide sequence listed in Table 5 or a sequencethat is at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at leastabout 99% identical to a polynucleotide sequence listed in Table 5.Acetolactate reductases capable of converting acetolactate to DHMB canbe identified, for example, by screening genetically altered yeast forchanges in acetolactate consumption, changes in DHMB production, changesin DHIV production, or changes in other downstream product (e.g.,butanol) production.

One way of identifying a gene involved in DHMB production comprisesmeasuring the amount of DHMB produced by individual yeast strains in ayeast knock-out library. Knock-out libraries are available, for example,from Open Biosystems® (a division of Thermo Fisher Scientific, Waltham,Mass.). In this method, a decrease in DHMB production indicates that thegene that has been knocked-out functions to increase DHMB production,and an increase in DHMB production indicates that the gene that has beenknocked-out functions to decrease DHMB production.

Two ways that a knockout (“KO”) library can be used to identifycandidate genes for involvement in DHMB synthesis include: (1) DHMB andDHIV accumulated in the culture during growth from endogenous substrates(acetolactate and NADPH or NADH) can be analyzed in samples fromcultures. These samples can be placed in a hot (80-100° C.) water bathfor 10-20 min, or diluted into a solution such as 2% formic acid thatwill kill and permeabilize the cells. After either treatment, smallmolecules will be found in the supernatant after centrifugation (5 min,1100×g). The DHMB/DHIV ratio of a control strain (e.g., BY4743) can becompared to that of the different KO derivatives, and the gene(s)missing from any strain(s) with exceptionally low DHMB/DHIV ratios canencode acetolactate reductase (ALR). (2) DHMB and/or DHIV formationrates in vitro from exogenous substrates (acetolactate and NADH and/orNADPH) can be measured in timed samples taken from a suspension ofpermeabilized cells, and inactivated in either of the ways describedabove. Since the substrates for DHMB and DHIV synthesis are the same,this allows one to measure the relative levels of ALR and KARI activityin the sample.

Another way of identifying a gene involved in DHMB production comprisesmeasuring the amount of DHMB produced by individual yeast strains in ayeast overexpression library. Overexpression libraries are available,for example, from Open Biosystems® (a division of Thermo FisherScientific, Waltham, Mass.). In this method, a decrease in DHMBproduction indicates that the overexpressed gene functions to decreaseDHMB production, and an increase in DHMB production indicates that theoverexpressed gene functions to increase DHMB production.

Another way of identifying a gene involved in DHMB production is tobiochemically analyze a DHMB-producing yeast strain. For example,DHMB-producing cells can be disrupted. This disruption can be performedat low pH and cold temperatures. The cell lysates can be separated intofractions, e.g., by adding ammonium sulfate or other techniques known tothose of skill in the art, and the resulting fractions can be assayedfor enzymatic activity. For example, the fractions can be assayed forthe ability to convert acetolactate to DHMB. Fractions with enzymaticactivity can be treated by methods known in the art to purify andconcentrate the enzyme (e.g., dialysis and chromatographic separation).When a sufficient purity and concentration is achieved, the enzyme canbe sequenced, and the corresponding gene encoding the acetolactatereductase capable of converting acetolactate to DHMB can be identified.

Furthermore, since the reduction of acetolactate to DHMB occurs inyeast, but does not occur to the same extent in E. coli, acetolactatereductases that are expressed in yeast, but not expressed in E. coli,can be selected for screening. Selected enzymes can be expressed inyeast or other protein expression systems and screened for thecapability to convert acetolactate to DHMB.

Enzymes capable of catalyzing the conversion of acetolactate to DHMB canbe screened by assaying for acetolactate levels, by assaying for DHMBlevels, by assaying for DHIV levels, or by assaying for any of thedownstream products in the conversion of DHIV to butanol, includingisobutanol.

DHMB can be measured using any technique known to those of skill in theart. For example, DHMB can be separated and quantified by methods knownto those of skill in the art and techniques described in the Examplesprovided herein. For example, DHMB can be separated and quantified usingliquid chromatography-mass spectrometry, liquid chromatography-nuclearmagnetic resonance (NMR), thin-layer chromatography, and/or HPLC withUV/Vis detection.

In embodiments, selected acetolactate reductase polynucleotides, genesand/or polypeptides disclosed herein can be modified or disrupted. Manysuitable methods are known to those of ordinary skill in the art andinclude those described for aldehyde dehydrogenase (above).

The modification of acetolactate reductase in a recombinant host celldisclosed herein to reduce or eliminate acetolactate reductase activitycan be confirmed using methods known in the art. For example, thepresence or absence of an acetolactate reductase-encoding polynucleotidesequence can be determined using PCR screening. A decrease inacetolactate reductase activity can also be determined based on areduction in conversion of acetolactate to DHMB. A decrease inacetolactate reductase activity can also be determined based on areduction in DHMB production. A decrease in acetolactate reductaseactivity can also be determined based on an increase in butanolproduction.

Thus, in some embodiments, a yeast that is capable of producing butanolproduces no more than about 5 mM, about 4 mM, about 3 mM, to about 2 mM,about 1 mM, about 0.9 mM, about 0.8 mM, about 0.7 mM, about 0.6 mM,about 0.5 mM, about 0.4 mM or about 0.3 mM DHMB. In some embodiments, ayeast producing butanol produces no more than about 5 mM, about 4 mM,about 3 mM, about 2 mM, about 1 mM, about 0.9 mM, about 0.8 mM., about0.7 mM, about 0.6 mM, about 0.5 mM, about 0.4 mM or about 0.3 mM DHMB.In some embodiments, a yeast producing butanol produces no more thanabout 0.2 mM or 0.2 mM DHMB.

In some embodiments, a yeast capable of producing butanol produces nomore than about 10 mM DHMB when cultured under fermentation conditionsfor at least about 50 hours. In some embodiments, a yeast capable ofproducing butanol produces no more than about 5 mM DHMB when culturedunder fermentation conditions for at least about 20 hours, at leastabout 25 hours, at least about 30 hours, at least about 35 hours, atleast about 40 hours, at least about 45 hours, or at least about 50hours. In some embodiments, a yeast capable of producing butanolproduced no more than about 3 mM DHMB when cultured under fermentationconditions for at least about 5 hours, at least about 10 hours, at leastabout 15 hours, at least about 20 hours, at least about 25 hours, atleast about 30 hours, at least about 35 hours, at least about 40 hours,at least about 45 hours, or at least about 50 hours.

In some embodiments, a yeast capable of producing butanol produced nomore than about 1 mM DHMB when cultured under fermentation conditionsfor at least about 1 hour, at least about 5 hours, at least about 10hours, at least about 15 hours, at least about 20 hours, at least about25 hours, at least about 30 hours, at least about 35 hours, at leastabout 40 hours, at least about 45 hours, or at least about 50 hours. Insome embodiments, a yeast capable of producing butanol produced no morethan about 0.5 mM DHMB when cultured under fermentation conditions forat least about 1 hour, at least about 5 hours, at least about 10 hours,at least about 15 hours, at least about 20 hours, at least about 25hours, at least about 30 hours, at least about 35 hours, at least about40 hours, at least about 45 hours, or at least about 50 hours.

In some embodiments, a yeast comprising at least one deletion, mutation,and/or substitution in an endogenous polynucleotide encoding anacetolactate reductase produces no more than about 0.5 times, about 0.4times, about 0.3 times, about 0.2 times, about 0.1 times, about 0.05times the amount of DHMB produced by a yeast containing the endogenouspolynucleotide encoding an acelotacatate reductase when cultured underfermentation conditions for the same amount of time.

In some embodiments, a yeast that is capable of producing butanolproduces an amount of DHIV that is at least about 5 mM, at least about 6mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or atleast about 10 mM.

In some embodiments, a yeast that is capable of producing butanolproduces an amount of DHIV that is at least about the amount of DHMBproduced. In some embodiments, a yeast that is capable of producingbutanol produces an amount of DHIV that is at least about twice, aboutthree times, about five times, about ten times, about 15 times, about 20times, about 25 times, about 30 times, about 35 times, about 40 times,about 45 times, or about 50 times the amount of DHMB produced.

In some embodiments, a yeast that is capable of producing butanolproduces DHIV at a rate that is at least about equal to the rate of DHMBproduction. In some embodiments, a yeast that is capable of producingbutanol produces DHIV at a rate that is at least about twice, aboutthree times, about five times, about ten times, about 15 times, about 20times, about 25 times, about 30 times, about 35 times, about 40 times,about 45 times, or about 50 times the rate of DHMB production.

In some embodiments, a yeast that is capable of producing butanolproduces less than 0.010 moles of DHMB per mole of glucose consumed. Insome embodiments, a yeast produces less than about 0.009, less thanabout 0.008, less than about 0.007, less than about 0.006, or less thanabout 0.005 moles of DHMB per mole of glucose consumed. In someembodiments, a yeast produces less than about 0.004, less than about0.003, less than about 0.002, or less than about 0.001 moles of DHMB permole of glucose consumed.

In some embodiments, acetolactate reductase activity is inhibited bychemical means. For example, acetolactate reductase could be inhibitedusing other known substrates such as those listed in Fujisawa et al.including L-serine, D-serine, 2-methyl-DL-serine, D-threonine,L-allothreonine, L-3-hydroxyisobutyrate, D-3-hydroxyisobutyrate,3-hydroxypropionate, L-3-hydroxybutyrate, and D-3-hydroxybutyrate(Biochimica et Biophysica Acta 1645:89-94 (2003), which is hereinincorporated by reference in its entirety).

DHMB Removal

In other embodiments described herein, a reduction in DHMB can beachieved by removing DHMB from a fermentation. Thus, fermentations withreduced DHMB concentrations are also described herein. Removal of DHMBcan result, for example, in a product of greater purity, or a productrequiring less processing to achieve a desired purity. Therefore,compositions comprising products of biosynthetic pathways such asethanol or butanol with increased purity are also provided.

DHMB can be removed during or after a fermentation process and can beremoved by any means known in the art. DHMB can be removed, for example,by extraction into an organic phase or reactive extraction.

In some embodiments, the fermentation broth comprises less than about0.5 mM DHMB. In some embodiments, the fermentation broth comprises lessthan about 1.0 mM DHMB after about 5 hours, about 10 hours, about 15hours, about 20 hours, about 25 hours, about 30 hours, about 35 hours,about 40 hours, about 45 hours, or about 50 hours of fermentation. Insome embodiments, the fermentation broth comprises less than about 5.0mM DHMB after about 20 hours, about 25 hours, about 30 hours, about 35hours, about 40 hours, about 45 hours, or about 50 hours offermentation.

Biosynthetic Pathways

While the KARI variants presented herein are believed to be suitable forproduction of isobutanol, it is envisioned that KARIs disclosed hereinmay be useful in any biosynthetic pathway which employs a substrate toproduct conversion catalyzed by KARI activity such as acetolactate to2,3-dihydroxyisovalerate or 2-aceto-2-hydroxybutanoate to2,3-dihydroxy-3-methylpentanoate. Such pathways include, but are notlimited to, pathways for producing pantothenic acid, valine, leucine,isoleucine or 3,3-dimethylmalate.

Certain suitable isobutanol biosynthetic pathways are disclosed in U.S.Pat. Nos. 7,851,188 and 7,993,889, each of which is incorporated byreference in their entireties herein. A diagram of the disclosedisobutanol biosynthetic pathways is provided in FIG. 1. As described inU.S. Pat. No. 7,851,188, steps in an example isobutanol biosyntheticpathway include conversion of:

-   -   pyruvate to acetolactate (see FIG. 1, pathway step a therein),        as catalyzed for example by acetolactate synthase (ALS),    -   acetolactate to 2,3-dihydroxyisovalerate (see FIG. 1, pathway        step b therein) as catalyzed for example by acetohydroxy acid        isomeroreductase (KARI);    -   2,3-dihydroxyisovalerate to 2-ketoisovalerate (see FIG. 1,        pathway step c therein) as catalyzed for example by acetohydroxy        acid dehydratase, also called dihydroxy-acid dehydratase (DHAD);    -   2-ketoisovalerate to isobutyraldehyde (see FIG. 1, pathway step        d therein) as catalyzed for example by branched-chain 2-keto        acid decarboxylase; and    -   isobutyraldehyde to isobutanol (see FIG. 1, pathway step e        therein) as catalyzed for example by branched-chain alcohol        dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thefollowing substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by ketol-acid reductoisomerase (KARI);    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by dihydroxyacid dehydratase;    -   α-ketoisovalerate to valine, which may be catalyzed, for        example, by transaminase or valine dehydrogenase;    -   valine to isobutylamine, which may be catalyzed, for example, by        valine decarboxylase;    -   isobutylamine to isobutyraldehyde, which may be catalyzed by,        for example, omega transaminase; and,    -   isobutyraldehyde to isobutanol, which may be catalyzed, for        example, by a branched-chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thefollowing substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by acetohydroxy acid reductoisomerase;    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by acetohydroxy acid dehydratase;    -   α-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for        example, by branched-chain keto acid dehydrogenase;    -   isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for        example, by acetylating aldehyde dehydrogenase; and,    -   isobutyraldehyde to isobutanol, which may be catalyzed, for        example, by a branched-chain alcohol dehydrogenase.

In another embodiment, the isobutanol biosynthetic pathway comprises thesubstrate to product conversions shown as steps k, g, and e in FIG. 1.

In another embodiment, the pathway comprising the substrate to productconversion catalyzed by KARI is a pantothenic acid biosynthetic pathwaycomprising the following substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by ketol-acid reductoisomerase (KARI);    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by dihydroxyacid dehydratase (DHAD);    -   α-ketoisovalerate to 2-dehydropantoate, which may be catalyzed,        for example, by 3-methyl-2-oxobutanoate hydroxymethyltransferase        (panB; which may be classified as EC 2.1.2.11);    -   2-dehydropantoate to (R)-pantoate, which may be catalyzed, for        example by 2-dehydropantoate 2-reductase (panE; which may be        classified as EC 1.1.1.169)    -   (R)-pantoate to (R)-pantothenate. which may be catalyzed, for        example, by pantoate-beta-alanine ligase (panC; which may be        classified as EC 6.3.2.1).

In another embodiment, the pathway comprising a substrate to productconversion catalyzed by KARI is a valine biosynthetic pathway comprisingthe following substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by ketol-acid reductoisomerase (KARI);    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by dihydroxyacid dehydratase (DHAD);    -   α-ketoisovalerate to valine, which may be catalyzed, for        example, by branched chain aminotransferase (ilvE (BAT); which        may be classified as EC 2.6.1.42).

In another embodiment, the pathway comprising a substrate to productconversion catalyzed by KARI is an isoleucine biosynthetic pathwaycomprising the following substrate to product conversions:

-   -   pyruvate and α-ketobutyrate to 2-aceto-2-hydroxybutanoate, which        may be catalyzed for example, by acetolactate synthase;    -   2-aceto-2-hydroxybutanoate to 2,3-dihydroxy-3-methylpentanoate,        which may be catalyzed for example, by KARI;    -   2,3-dihydroxy-3-methylpentanoate to 3-methyl-2-oxo-pentanoate,        which may be catalyzed for example, by DHAD;    -   3-methyl-2-oxo-pentanoate to isoleucine, which may be catalyzed,        for example, by branched chain aminotransferase (ilvE (BAT);        which may be classified as EC 2.6.1.42).

In another embodiment, the pathway comprising a substrate to productconversion catalyzed by KARI is a leucine biosynthetic pathwaycomprising the following substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by ketol-acid reductoisomerase (KARI);    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by dihydroxyacid dehydratase (DHAD);    -   α-ketoisovalerate to 2-isopropylmalate, which may be catalyzed,        for example, by 2-isopropylmalate synthase (leuA, which may be        classified as EC 2.3.3.13);    -   2-isopropylmalate to 2-isopropylmaleate, which may be catalyzed,        for example, by 3-isopropylmalate dehydratase (leu1; which may        be classified as EC 4.2.1.33);    -   2-isopropylmaleate to 3-isopropylmalate, which may be catalyzed,        for example, by 3-isopropylmalate dehydratase (leu1; which may        be classified as EC 4.2.1.33);    -   3-isopropylmalate to 2-isopropyl-3-oxosuccinate, which may be        catalyzed, for example by 3-isopropylmalate dehydrogenase (leuB;        which may be classified as EC 1.1.1.85);    -   2-isopropyl-3-oxosuccinate to 4-methyl-2-oxopentanoate        (spontaneous reaction); and    -   4-methyl-2-oxopentanoate to leucine, which may be catalyzed, for        example, by branched chain aminotransferase (ilvE (BAT); which        may be classified as EC 2.6.1.42)

In another embodiment, the pathway comprising a substrate to productconversion catalyzed by KARI is a 3,3-dimethylmalate biosyntheticpathway comprising the following substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,        by acetolactate synthase;    -   acetolactate to 2,3-dihydroxyisovalerate, which may be        catalyzed, for example, by ketol-acid reductoisomerase (KARI);    -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be        catalyzed, for example, by dihydroxyacid dehydratase (DHAD);    -   α-ketoisovalerate to (R)-3,3 dimethylmalate, which may be        catalyzed for example, by dimethylmalatedehydrogenase (DMMD;        which may be classified as 1.1.1.84).

Genes and polypeptides that can be used for certain of the substrate toproduct conversions described above as well as those for additionalisobutanol pathways, are available in the art. For example, described inU.S. Patent Appl. Pub. No. 2007/0092957 and PCT Pub. No. WO 2011/019894,both incorporated by reference in their entireties herein. US Appl. Pub.Nos. 2011/019894, 2007/0092957, 2010/0081154, which are hereinincorporated by reference in their entireties, describe dihydroxyaciddehydratases including those from Lactococcus lactis and Streptococcusmutans. Ketoisovalerate decarboxylases include those derived fromLactococcus lactis, Macrococcus caseolyticus (SEQ ID NO: 171) andListeria grayi (SEQ ID NO: 170). U.S. Patent Appl. Publ. No.2009/0269823 and U.S. Appl. Publ. No. 2011/0269199, incorporated byreference in their entireties, describe alcohol dehydrogenases,including those that utilize NADH as a cofactor. Alcohol dehydrogenasesinclude SadB from Achromobacter xylosoxidans. Additional alcoholdehydrogenases include horse liver ADH and Beijerinkia indica ADH.Alcohol dehydrogenases include those that utilize NADH as a cofactor. Inone embodiment an isobutanol biosynthetic pathway comprises a) aketol-acid reductoisomerase that has a K_(M) for NADH less than about300 μM, less than about 100 μM, less than about 50 μM, less than about20 μM or less than about 10 μM; b) an alcohol dehydrogenase thatutilizes NADH as a cofactor; or c) both a) and b).

WO 2011/019894 and U.S. Appl. Pub. Nos. 2011/019894, 2007/0092957,2010/0081154, which are herein incorporated by reference in theirentireties, describe suitable dihydroxyacid dehydratases. Methods ofincreasing DHAD activity are described, for example, in U.S. Appl. Pub.Nos. 2010/0081173 and 2012/0064561A1, which are herein incorporated byreference in their entireties.

Additional genes that can be used can be identified by one skilled inthe art through bioinformatics or using methods well-known in the art.

Additionally described in U.S. Appl. Pub. No. US 2007/0092957 A1, whichis incorporated by reference herein in its entirety, are construction ofchimeric genes and genetic engineering of bacteria and yeast forisobutanol production using the disclosed isobutanol biosyntheticpathways. Such construction and engineering methods could be readilyemployed by those of ordinary skill in the art to construct otherpathways disclosed herein and/or known in the art which include asubstrate to product conversion catalyzed by KARI activity.

Suitable ketol-acid reductoisomerase (KARI) enzymes are describedelsewhere herein. In some embodiments, the KARI enzyme has a specificactivity of at least about 0.1 micromoles/min/mg, at least about 0.2micromoles/min/mg, at least about 0.3 micromoles/min/mg, at least about0.4 micromoles/min/mg, at least about 0.5 micromoles/min/mg, at leastabout 0.6 micromoles/min/mg, at least about 0.7 micromoles/min/mg, atleast about 0.8 micromoles/min/mg, at least about 0.9 micromoles/min/mg,at least about 1.0 micromoles/min/mg, or at least about 1.1micromoles/min/mg. Suitable polypeptides to catalyze the substrate toproduct conversion acetolactate to 2,3-dihydroxyisovalerate includethose that that have a K_(M) for NADH less than about 300 μM, less thanabout 100 μM, less than about 50 μM, less than about 25 μM or less thanabout 10 μM.

Modifications

Functional deletion of the pyruvate decarboxylase gene has been used toincrease the availability of pyruvate for utilization in biosyntheticproduct pathways. For example, U.S. Application Publication No. US2007/0031950 A1, which is herein incorporated by reference in itsentirety, discloses a yeast strain with a disruption of one or morepyruvate decarboxylase genes and expression of a D-Iactate dehydrogenasegene, which is used for production of D-lactic acid. U.S. Appl. Pub. No.U.S. 2005/0059136 A1, which is herein incorporated by reference in itsentirety, discloses glucose tolerant two carbon source independent(GCSI) yeast strains with no pyruvate decarboxylase activity, which canhave an exogenous lactate dehydrogenase gene. Nevoigt and Stahl (Yeast12:1331-1337 (1996)) describe the impact of reduced pyruvatedecarboxylase and increased NAD-dependent glycerol-3-phosphatedehydrogenase in Saccharomyces cerevisiae on glycerol yield. U.S. Appl.Pub. No. 2009/0305363, which is herein incorporated by reference in itsentirety, discloses increased conversion of pyruvate to acetolactate byengineering yeast for expression of a cytosol-localized acetolactatesynthase and substantial elimination of pyruvate decarboxylase activity.

In embodiments of the invention, a recombinant host cell disclosedherein can comprise a modification in an endogenous polynucleotideencoding a polypeptide having pyruvate decarboxylase (PDC) activity or amodification in an endogenous polypeptide having PDC activity. Inembodiments, a recombinant host cell disclosed herein can have amodification or disruption of a polynucleotide, gene and/or polypeptideencoding PDC. In embodiments, a recombinant host cell comprises adeletion, mutation, and/or substitution in an endogenous polynucleotideor gene encoding a polypeptide having PDC activity, or in an endogenouspolypeptides having PDC activity. Such modifications, disruptions,deletions, mutations, and/or substitutions can result in PDC activitythat is reduced or eliminated, resulting, for example, in a PDCknock-out (PDC-KO) phenotype.

In embodiments of the invention, an endogenous pyruvate decarboxylaseactivity of a recombinant host cell disclosed herein converts pyruvateto acetaldehyde, which can then be converted to ethanol or to acetyl-CoAvia acetate. In other embodiments, a recombinant host cell isKluyveromyces lactis containing one gene encoding pyruvatedecarboxylase, Candida glabrata containing one gene encoding pyruvatedecarboxylase, or Schizosaccharomyces pombe containing one gene encodingpyruvate decarboxylase.

In other embodiments, a recombinant host cell is Saccharomycescerevisiae containing three isozymes of pyruvate decarboxylase encodedby the PDC1, PDC5, and PDC6 genes, as well as a pyruvate decarboxylaseregulatory gene, PDC2. In a non-limiting example in S. cerevisiae, thePDC1 and PDC5 genes, or the PDC1, PDC5, and PDC6 genes, are disrupted.In another non-limiting example in S. cerevisiae, pyruvate decarboxylaseactivity can be reduced by disrupting the PDC2 regulatory gene. Inanother non-limiting example in S. cerevisiae, polynucleotides or genesencoding pyruvate decarboxylase proteins such as those having about 70%to about 75%, about 75% to about 80%, about 80% to about 85%, about 85%to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%,or about 99% sequence identity to PDC1, PDC2, PDC5 and/or PDC6 can bedisrupted.

In embodiments, a polypeptide having PDC activity or a polynucleotide orgene encoding a polypeptide having PDC activity corresponds to EnzymeCommission Number EC 4.1.1.1. In other embodiments, a PDC gene of arecombinant host cell disclosed herein is not active under thefermentation conditions used, and therefore such a gene would not needto be modified or inactivated.

Examples of recombinant host cells with reduced pyruvate decarboxylaseactivity due to disruption of pyruvate decarboxylase encoding genes havebeen reported, such as for Saccharomyces in Flikweert et al. (Yeast(1996) 12:247-257), for Kluyveromyces in Bianchi et al. (Mol. Microbiol.(1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann(Mol. Gen. Genet. (1993) 241:657-666). Saccharomyces strains having nopyruvate decarboxylase activity are available from the ATCC withAccession #200027 and #200028. Examples of PDC polynucleotides, genesand/or polypeptides that can be targeted for modification orinactivation in the recombinant host cells disclosed herein include, butare not limited to, those of the following Table 6.

TABLE 6 Pyruvate decarboxylase target gene coding regions and proteins.SEQ ID NO: SEQ ID NO: Description Nucleic acid Amino acid PDC1 pyruvatedecarboxylase from 234 235 Saccharomyces cerevisiae PDC5 pyruvatedecarboxylase from 236 237 Saccharomyces cerevisiae PDC6 pyruvatedecarboxylase from 238 239 Saccharomyces cerevisiae pyruvatedecarboxylase from 240 241 Candida glabrata PDC1 pyruvate decarboxylasefrom 242 243 Pichia stipitis PDC2 pyruvate decarboxylase from 244 245Pichia stipitis pyruvate decarboxylase from 246 247 Kluyveromyces lactispyruvate decarboxylase from 248 249 Yarrowia lipolytica pyruvatedecarboxylase from 250 251 Schizosaccharomyces pombe pyruvatedecarboxylase from 252 253 Zygosaccharomyces rouxii

Other examples of PDC polynucleotides, genes and polypeptides that canbe targeted for modification or inactivation in a recombinant host celldisclosed herein include, but are not limited to, PDC polynucleotides,genes and/or polypeptides having at least about 70% to about 75%, about75% to about 80%, about 80% to about 85%, about 85% to about 90%, about90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequenceidentity to any one of the sequences of Table 6, wherein such apolynucleotide or gene encodes, or such a polypeptide has, PDC activity.Still other examples of PDC polynucleotides, genes and polypeptides thatcan be targeted for modification or inactivation in a recombinant hostcell disclosed herein include, but are not limited to an active variant,fragment or derivative of any one of the sequences of Table 6, whereinsuch a polynucleotide or gene encodes, or such a polypeptide has, PDCactivity.

In embodiments, a polynucleotide, gene and/or polypeptide encoding a PDCsequence disclosed herein or known in the art can be modified, asdisclosed above for aldehyde dehydrogenase. In other embodiments, apolynucleotide, gene and/or polypeptide encoding PDC can be used toidentify another PDC polynucleotide, gene and/or polypeptide sequence orto identify a PDC homolog in other cells, as disclosed above foracetolactate dehydrogenase. Such a PDC encoding sequence can beidentified, for example, in the literature and/or in bioinformaticsdatabases well known to the skilled person. For example, theidentification of a PDC encoding sequence in other cell types usingbioinformatics can be accomplished through BLAST (as described above)searching of publicly available databases with a known PDC encoding DNAand polypeptide sequence, such as those provided herein. Identities arebased on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix.

The modification of PDC in a recombinant host cell disclosed herein toreduce or eliminate PDC activity can be confirmed using methods known inthe art. For example, disruption of a particular pyruvate decarboxylasecould be confirmed with PCR screening using primers external to the genesequence, or by Southern blot using a probe designed to the pyruvatedecarboxylase gene sequence. Alternatively, one could utilize analyticalmethods such as gas chromatography or HPLC to screen strains fordecreased or eliminated production of acetaldehyde and/or ethanol.

Functional deletion of the hexokinase 2 gene has been used to reduceglucose repression and to increase the availability of pyruvate forutilization in biosynthetic pathways. For example, InternationalPublication No. WO 2000/061722 A1, which is incorporated herein byreference in its entirety discloses the production of yeast biomass byaerobically growing yeast having one or more functionally deletedhexokinase 2 genes or analogs. In addition, Rossell et al. (YeastResearch 8:155-164 (2008)) found that Saccharomyces cerevisiae with adeletion of the hexokinase 2 gene showed 75% reduction in fermentativecapacity, defined as the specific rate of carbon dioxide productionunder sugar-excess and anaerobic conditions. After starvation, thefermentation capacity was similar to that of a strain without thehexokinase 2 gene deletion. Diderich et al. (Applied and EnvironmentalMicrobiology 67:1587-1593 (2001)) found that S. cerevisiae with adeletion of the hexokinase 2 gene had lower pyruvate decarboxylaseactivity.

In embodiments, a recombinant host cell disclosed herein can comprise amodification in an endogenous polynucleotide encoding a polypeptidehaving hexokinase 2 activity and/or a modification in a polypeptidehaving hexokinase 2 activity. In embodiments, a recombinant host celldisclosed herein can have a modification or disruption of apolynucleotide, gene or polypeptide encoding hexokinase 2. Inembodiments, a recombinant host cell comprises a deletion, mutation,and/or substitution in an endogenous polynucleotide or gene encoding apolypeptide having hexokinase 2 activity, or an endogenous polypeptidehaving hexokinase 2 activity. Such modifications, disruptions,deletions, mutations, and/or substitutions can result in hexokinase 2activity that is reduced or eliminated, resulting, for example, in ahexokinase 2 knockout (HXK2-KO) phenotype. In embodiments, the host cellcomprises a modification as described in U.S. Appn. Serial. Nos.2011/0124060 A1 or 2012/0015416 A1, which are incorporated herein byreference in their entireties.

In embodiments, a polypeptide having hexokinase 2 activity can catalyzethe conversion of hexose to hexose-6-phosphate, and/or can catalyze theconversion of D-glucose to D-glucose 6-phosphate, D-fructose toD-fructose 6-phosphate, and/or D-mannose to D-mannose 6-phosphate. Inother embodiments, a polynucleotide, gene or polypeptide havinghexokinase 2 activity can correspond to Enzyme Commission Number EC2.7.1.1.

In embodiments of the invention, a recombinant host cell can be S.cerevisiae and a polynucleotide, gene or polypeptide having hexokinase 2activity can be HXK2. In other embodiments, a recombinant host cell canbe K. lactis and a polynucleotide, gene or polypeptide having hexokinase2 activity can be RAGS. In other embodiments, a recombinant host cellcan be H. polymorpha and a polynucleotide, gene or polypeptide havinghexokinase 2 activity can be HPGLKI. In other embodiments, a recombinanthost cell can be S. pombe and a polynucleotide, gene or polypeptidehaving hexokinase 2 activity can be HXK2.

Examples of hexokinase 2 polynucleotides, genes and polypeptides thatcan be targeted for modification or inactivation in a recombinant hostcell disclosed herein include, but are not limited to, those of thefollowing Table 7.

TABLE 7 Hexokinase 2 target gene coding regions and proteins. HXK2 fromS. cerevisiae Nucleic acid (SEQ ID NO: 254) Amino acid (SEQ ID NO: 255)RAG5 from K. lactis Nucleic acid (SEQ ID NO: 256) Amino acid (SEQ ID NO:257) HPGLK1 from H. polymorpha Nucleic acid (SEQ ID NO: 258) Amino acid(SEQ ID NO: 259) HXK2 from S. pombe Nucleic acid (SEQ ID NO: 260) Aminoacid (SEQ ID NO: 261)

Other examples of hexokinase 2 polynucleotides, genes and polypeptidesthat can be targeted for modification or inactivation in a recombinanthost cell disclosed herein include, but are not limited to, hexokinase 2polynucleotides, genes and/or polypeptides having at least about 70% toabout 75%, about 75% to about 80%, about 80% to about 85%, about 85% toabout 90%, about 90% to about 95%, about 96%, about 97%, about 98%, orabout 99% sequence identity to any one of the sequences of Table 9,wherein such a polynucleotide or gene encodes, or such a polypeptidehas, hexokinase 2 activity. Still other examples of hexokinase 2polynucleotides, genes and polypeptides that can be targeted formodification or inactivation in a recombinant host cell disclosed hereininclude, but are not limited to an active variant, fragment orderivative of any one of the sequences of Table 7, wherein such apolynucleotide or gene encodes, or such a polypeptide has, hexokinase 2activity.

In embodiments, a polynucleotide, gene and/or polypeptide encoding ahexokinase 2 sequence disclosed herein or known in the art can bemodified or disrupted, as disclosed above for aldehyde dehydrogenase. Inother embodiments, a polynucleotide, gene and/or polypeptide encodinghexokinase 2 can be used to identify another hexokinase 2polynucleotide, gene and/or polypeptide sequence or to identify ahexokinase 2 homolog in other cells, as disclosed above for aldehydedehydrogenase. Such a hexokinase 2 encoding sequence can be identified,for example, in the literature and/or in bioinformatics databases wellknown to the skilled person. For example, the identification of ahexokinase 2 encoding sequence in other cell types using bioinformaticscan be accomplished through BLAST (as described above) searching ofpublicly available databases with a known hexokinase 2 encoding DNA andpolypeptide sequence, such as those provided herein. Identities arebased on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix.

The modification of hexokinase 2 in a recombinant host cell disclosedherein to reduce or eliminate hexokinase 2 activity can be confirmedusing methods known in the art. For example, disruption of hexokinase 2could be confirmed with PCR screening using primers external to thehexokinase 2 gene, or by Southern blot using a probe designed to thehexokinase 2 gene sequence. Alternatively, one could examine putativehexokinase 2 knockout strains for increased biomass yield onglucose-containing media.

Examples of additional modifications that can be useful in cellsprovided herein include modifications to reduce glycerol-3-phosphatedehydrogenase activity and/or disruption in at least one gene encoding apolypeptide having pyruvate decarboxylase activity or a disruption in atleast one gene encoding a regulatory element controlling pyruvatedecarboxylase gene expression as described in U.S. Patent Appl. Pub. No.2009/0305363 (incorporated herein by reference in its entirety),modifications to a host cell that provide for increased carbon fluxthrough an Entner-Doudoroff Pathway or reducing equivalents balance asdescribed in U.S. Patent Appl. Pub. No. 2010/0120105 (incorporatedherein by reference in its entirety). Other modifications includeintegration of at least one polynucleotide encoding a polypeptide thatcatalyzes a step in a pyruvate-utilizing biosynthetic pathway describedin PCT Appn. Pub. No. WO 2012/033832, which is herein incorporated byreference in its entirety. A genetic modification which has the effectof reducing glucose repression wherein the yeast production host cell ispdc—is described in U.S. Appl. Publ No. US 2011/0124060, which is hereinincorporated by reference in its entirety.

U.S. Appl. Publ. No. 20120064561A1, which is herein incorporated byreference in its entirety, discloses recombinant host cells comprising(a) at least one heterologous polynucleotide encoding a polypeptidehaving dihydroxy-acid dehydratase activity: and (b)(i) at least onedeletion, mutation, and/or substitution in an endogenous gene encoding apolypeptide affecting Fe—S cluster biosynthesis; and/or (ii) at leastone heterologous polynucleotide encoding a polypeptide affecting Fe—Scluster biosynthesis. In embodiments, the polypeptide affecting Fe—Scluster biosynthesis is encoded by AFT1, AFT2, FRA2, GRX3, or CCC1 Inembodiments, the polypeptide affecting Fe—S cluster biosynthesis isconstitutive mutant AFT1 L99A, AFT1 L102A, AFT1 C291F, or AFT1 C293F.

Additionally, host cells can comprise heterologous polynucleotidesencoding a polypeptides with phosphoketolase activity and/or aheterologous polynucleotide encoding a polypeptide withphosphotransacetylase activity such as, for example, those encoded bySEQ ID NOs: 262 and 263, and as described in PCT Appn. Pub. No. WO2011/159853 and U.S. Appl. Publ. No. 20120156735A1, which are hereinincorporated by reference in their entireties.

Microbial Hosts for Isobutanol Production

Microbial hosts for isobutanol production can be selected from bacteria,cyanobacteria, filamentous fungi and yeasts. The microbial host used forbutanol production should be tolerant to isobutanol so that the yield isnot limited by butanol toxicity. Although butanol-tolerant mutants havebeen isolated from solventogenic Clostridia, little information isavailable concerning the butanol tolerance of other potentially usefulbacterial strains. Most of the studies on the comparison of alcoholtolerance in bacteria suggest that butanol is more toxic than ethanol(de Cavalho, et al., Microsc. Res. Tech., 64: 215-22, 2004) and(Kabelitz, et at, FEMS Microbiol. Lett., 220: 223-227, 2003, Tomas, etal., J. Bacteriol., 186: 2006-2018, 2004) report that the yield of1-butanol during fermentation in Clostridium acetobutylicum can belimited by 1-butanol toxicity. The primary effect of 1-butanol onClostridium acetobutylicum is disruption of membrane functions (Hermannet al., Appl. Environ. Microbial., 50: 1238-1243, 1985).

The microbial hosts selected for the production of isobutanol should betolerant to isobutanol and should be able to convert carbohydrates toisobutanol. The criteria for selection of suitable microbial hostsinclude the following: intrinsic tolerance to isobutanol, high rate ofglucose utilization, availability of genetic tools for genemanipulation, and the ability to generate stable chromosomalalterations.

Suitable host strains with a tolerance for isobutanol can be identifiedby screening based on the intrinsic tolerance of the strain. Theintrinsic tolerance of microbes to isobutanol can be measured bydetermining the concentration of isobutanol that is responsible for 50%inhibition of the growth rate (IC50) when grown in a minimal medium. TheIC50 values can be determined using methods known in the art. Forexample, the microbes of interest can be grown in the presence ofvarious amounts of isobutanol and the growth rate monitored by measuringthe optical density at 600 nanometers. The doubling time can becalculated from the logarithmic part of the growth curve and used as ameasure of the growth rate. The concentration of isobutanol thatproduces 50% inhibition of growth can be determined from a graph of thepercent inhibition of growth versus the isobutanol concentration. In oneembodiment, the host strain has an IC₅₀ for isobutanol of greater thanabout 0.5%.

The microbial host for isobutanol production should also utilize glucoseat a high rate. Most microbes are capable of metabolizing carbohydrates.However, certain environmental microbes cannot metabolize carbohydratesto high efficiency, and therefore would not be suitable hosts.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. The mode of gene transfertechnology can be by electroporation, conjugation, transduction ornatural transformation. A broad range of host conjugative plasmids anddrug resistance markers are available. The cloning vectors are tailoredto the host microorganisms based on the nature of antibiotic resistancemarkers that can function in that host.

The microbial host also has to be manipulated in order to inactivatecompeting pathways for carbon flow by deleting various genes. Thisrequires the availability of either transposons to direct inactivationor chromosomal integration vectors. Additionally, the production hostshould be amenable to chemical mutagenesis so that mutations to improveintrinsic isobutanol tolerance can be obtained.

Based on the criteria described above, suitable microbial hosts for theproduction of isobutanol include, but are not limited to, members of thegenera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Issatchenkia, Hansenula, Kluyveromyces,and Saccharomyces. Suitable hosts include: Escherichia coli, Alcaligeneseutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcusetythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcusfaecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillussubtilis and Saccharomyces cerevisiae. In some embodiments, the hostcell is Saccharomyces cerevisiae. S. cerevisiae yeast are known in theart and are available from a variety of sources, including, but notlimited to, American Type Culture Collection (Rockville, Md.),Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts,Martrex, and Lallemand. S. cerevisiae include, but are not limited to,BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio-Ferm® XRyeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand PotDistillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Greenyeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1,CBS7959, CBS7960, and CBS7961.

Construction of Production Host

Recombinant microorganisms containing the necessary genes that willencode the enzymatic pathway for the conversion of a fermentable carbonsubstrate to butanol can be constructed using techniques well known inthe art. In the present invention, genes encoding the enzymes of one ofthe isobutanol biosynthetic pathways of the invention, for example,acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxyacid dehydratase, branched-chain α-keto acid decarboxylase, andbranched-chain alcohol dehydrogenase, can be isolated from varioussources, as described above.

Methods of obtaining desired genes from a genome are common and wellknown in the art of molecular biology. For example, if the sequence ofthe gene is known, suitable genomic libraries can be created byrestriction endonuclease digestion and can be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA can be amplified using standard primer-directedamplification methods such as polymerase chain reaction (U.S. Pat. No.4,683,202) to obtain amounts of DNA suitable for transformation usingappropriate vectors. Tools for codon optimization for expression in aheterologous host are readily available. Some tools for codonoptimization are available based on the GC content of the hostmicroorganism.

Once the relevant pathway genes are identified and isolated they can betransformed into suitable expression hosts by means well known in theart. Vectors or cassettes useful for the transformation of a variety ofhost cells are common and commercially available from companies such asEPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.),Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly,Mass.). Typically the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. Both controlregions can be derived from genes homologous to the transformed hostcell, although it is to be understood that such control regions can alsobe derived from genes that are not native to the specific species chosenas a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant pathway coding regions in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genetic elements, including thoseused in the Examples, is suitable for the present invention including,but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH,ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression inSaccharomyces); AOX1 (useful for expression in Pichia); and lac, ara,tet, trp, IP_(L), IP_(R), T7, tac, and trc (useful for expression inEscherichia coli, Alcaligenes, and Pseudomonas) as well as the amy, apr,npr promoters and various phage promoters useful for expression inBacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans.For yeast recombinant host cells, a number of promoters can be used inconstructing expression cassettes for genes, including, but not limitedto, the following constitutive promoters suitable for use in yeast:FBA1, TDH3 (GPD), ADH1, ILV5, and GPM1; and the following induciblepromoters suitable for use in yeast: GAL1, GAL10, OLE1, and CUP1. Otheryeast promoters include hybrid promoters UAS(PGK1)-FBA1p (SEQ ID NO:264), UAS(PGK1)-ENO2p (SEQ ID NO: 265), UAS(FBA1)-PDC1p (SEQ ID NO:266), UAS(PGK1)-PDC1p (SEQ ID NO: 267), and UAS(PGK)-OLE1p (SEC) ID NO:268).

Promoters, transcriptional terminators, and coding regions can be clonedinto a yeast 2 micron plasmid and transformed into yeast cells (Ludwig,et al. Gene, 132: 33-40, 1993; US Appl. Pub. No. 20080261861A1).

Adjusting the amount of gene expression in a given host may be achievedby varying the level of transcription, such as through selection ofnative or artificial promoters. In addition, techniques such as the useof promoter libraries to achieve desired levels of gene transcriptionare well known in the art. Such libraries can be generated usingtechniques known in the art, for example, by cloning of random cDNAfragments in front of gene cassettes (Goh et al. (2002) AEM 99, 17025),by modulating regulatory sequences present within promoters (Ligr et al.(2006) Genetics 172, 2113), or by mutagenesis of known promotersequences (Alper et al. (2005) PNAS, 12678; Nevoigt et al. (2005) AEM72, 5266).

Termination control regions can also be derived from various genesnative to the hosts. Optionally, a termination site can be unnecessaryorcan be included.

Certain vectors are capable of replicating in a broad range of hostbacteria and can be transferred by conjugation. The complete andannotated sequence of pRK404 and three related vectors-pRK437, pRK442,and pRK442(H) are available. These derivatives have proven to bevaluable tools for genetic manipulation in Gram-negative bacteria (Scottet al., Plasmid, 50: 74-79, 2003). Several plasmid derivatives ofbroad-host-range Inc P4 plasmid RSF1010 are also available withpromoters that can function in a range of Gram-negative bacteria.Plasmid pAYC36 and pAYC37, have active promoters along with multiplecloning sites to allow for the heterologous gene expression inGram-negative bacteria.

Chromosomal gene replacement tools are also widely available. Forexample, a thermosensitive variant of the broad-host-range repliconpWV101 has been modified to construct a plasmid pVE6002 which can beused to effect gene replacement in a range of Gram-positive bacteria(Maguin et al., J. Bacteriol., 174: 5633-5638, 1992). Additionally, invitro transposomes are available to create random mutations in a varietyof genomes from commercial sources such as EPICENTRE®.

The expression of a butanol biosynthetic pathway in various microbialhosts is described in more detail below.

Expression of a Butanol Biosynthetic Pathway in E. Coli

Vectors or cassettes useful for the transformation of E. coli are commonand commercially available from the companies listed above. For example,the genes of an isobutanol biosynthetic pathway can be isolated fromvarious sources, cloned into a modified pUC19 vector and transformedinto E. coli NM522.

Expression of a Butanol Biosynthetic Pathway in Rhodococcus Erythropolis

A series of E. coli-Rhodococcus shuttle vectors are available forexpression in R. erythropolis, including, but not limited to, pRhBR17and pDA71 (Kostichka et al., Appl. Microbial. Biotechnol., 62: 61-68,2003). Additionally, a series of promoters are available forheterologous gene expression in R. erythropolis (Nakashima et al., Appl.Environ. Microbiol., 70: 5557-5568, 2004 and Tao et al., Appl.Microbiol. Biotechnol., 68: 346-354, 2005). Targeted gene disruption ofchromosomal genes in R. erythropolis can be created using the methoddescribed by Tao et al., supra, and Brans et al. (Appl. Environ.Microbiol., 66: 2029-2036, 2000).

The heterologous genes required for the production of isobutanol, asdescribed above, can be cloned initially in pDA71 or pRhBR71 andtransformed into E. coli. The vectors can then be transformed into R.erythropolis by electroporation, as described by Kostichka et al.,supra. The recombinants can be grown in synthetic medium containingglucose and the production of isobutanol can be followed using methodsknown in the art.

Expression of a Butanol Biosynthetic Pathway in B. Subtilis

Methods for gene expression and creation of mutations in B. subtilis arealso well known in the art. For example, the genes of an isobutanolbiosynthetic pathway can be isolated from various sources, cloned into amodified pUC19 vector and transformed into Bacillus subtilis BE1010.Additionally, the five genes of an isobutanol biosynthetic pathway canbe split into two operons for expression. The three genes of the pathway(bubB, ilvD, and kivD) can be integrated into the chromosome of Bacillussubtilis BE1010 (Payne, et al., J. Bacteriol., 173, 2278-2282, 1991).The remaining two genes (ilvC and bdhB) can be cloned into an expressionvector and transformed into the Bacillus strain carrying the integratedisobutanol genes

Expression of a Butanol Biosynthetic Pathway in B. Licheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtiliscan be used to transform B. licheniformis by either protoplasttransformation or electroporation. The genes required for the productionof isobutanol can be cloned in plasmids pBE20 or pBE60 derivatives(Nagarajan et al., Gene, 114: 121-126, 1992). Methods to transform B.licheniformis are known in the art (Fleming et al. Appl. Environ,Microbiol., 61: 3775-3780, 1995). The plasmids constructed forexpression in B. subtilis can be transformed into B. licheniformis toproduce a recombinant microbial host that produces isobutanol.

Expression of a Butanol Biosynthetic Pathway in Paenibacillus Macerans

Plasmids can be constructed as described above for expression in B.subtilis and used to transform Paenibacillus macerans by protoplasttransformation to produce a recombinant microbial host that producesisobutanol.

Expression of the Butanol Biosynthetic Pathway in Alcaligenes(Ralstonia) Eutrophus

Methods for gene expression and creation of mutations in Alcaligeneseutrophus are known in the art (Taghavi et al., Appl. Environ.Microbiol., 60: 3585-3591, 1994). The genes for an isobutanolbiosynthetic pathway can be cloned in any of the broad host rangevectors described above, and electroporated to generate recombinantsthat produce isobutanol. The poly(hydroxybutyrate) pathway inAlcaligenes has been described in detail, a variety of genetictechniques to modify the Alcaligenes eutrophus genome is known, andthose tools can be applied for engineering an isobutanol biosyntheticpathway.

Expression of a Butanol Biosynthetic Pathway in Pseudomonas putida

Methods for gene expression in Pseudomonas putida are known in the art(see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which isincorporated herein by reference). The butanol pathway genes can beinserted into pPCU18 and this ligated DNA can be electroporated intoelectrocompetent Pseudomonas putida DOT-T1 C5aAR1 cells to generaterecombinants that produce isobutanol.

Expression of a Butanol Biosynthetic Pathway in Saccharomyces cerevisiae

Methods for gene expression in Saccharomyces cerevisiae are known in theart (e.g., Methods in Enzymology, Volume 194, Guide to Yeast Geneticsand Molecular and Cell Biology, Part A, 2004, Christine Guthrie andGerald R. Fink, eds., Elsevier Academic Press, San Diego, Calif.).Expression of genes in yeast typically requires a promoter, followed bythe gene of interest, and a transcriptional terminator. A number ofyeast promoters, including those used in the Examples herein, can beused in constructing expression cassettes for genes encoding anisobutanol biosynthetic pathway, including, but not limited toconstitutive promoters FBA, GPD, ADH1, and GPM, and the induciblepromoters GAL1, GAL10, and CUP1. Suitable transcriptional terminatorsinclude, but are not limited to FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1,and ADH1. For example, suitable promoters, transcriptional terminators,and the genes of an isobutanol biosynthetic pathway can be cloned intoE. coli-yeast shuttle vectors and transformed into yeast cells asdescribed in U.S. App. Pub. No. 20100129886. These vectors allow strainpropagation in both E. coli and yeast strains. Typically the vectorcontains a selectable marker and sequences allowing autonomousreplication or chromosomal integration in the desired host. Typicallyused plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, andpRS426 (American Type Culture Collection, Rockville, Md.), which containan E. coli replication origin (e.g., pMB1), a yeast 2μ origin ofreplication, and a marker for nutritional selection. The selectionmarkers for these four vectors are His3 (vector pRS423), Trp1 (vectorpRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Construction ofexpression vectors with genes encoding polypeptides of interest can beperformed by either standard molecular cloning techniques in E. coli orby the gap repair recombination method in yeast.

The gap repair cloning approach takes advantage of the highly efficienthomologous recombination in yeast. Typically, a yeast vector DNA isdigested (e.g., in its multiple cloning site) to create a “gap” in itssequence. A number of insert DNAs of interest are generated that containa ≧21 bp sequence at both the 5′ and the 3′ ends that sequentiallyoverlap with each other, and with the 5′ and 3′ terminus of the vectorDNA. For example, to construct a yeast expression vector for “Gene X′, ayeast promoter and a yeast terminator are selected for the expressioncassette. The promoter and terminator are amplified from the yeastgenomic DNA, and Gene X is either PCR amplified from its source organismor obtained from a cloning vector comprising Gene X sequence. There isat least a 21 bp overlapping sequence between the 5′ end of thelinearized vector and the promoter sequence, between the promoter andGene X, between Gene X and the terminator sequence, and between theterminator and the 3′ end of the linearized vector. The “gapped” vectorand the insert DNAs are then co-transformed into a yeast strain andplated on the medium containing the appropriate compound mixtures thatallow complementation of the nutritional selection markers on theplasmids. The presence of correct insert combinations can be confirmedby PCR mapping using plasmid DNA prepared from the selected cells. Theplasmid DNA isolated from yeast (usually low in concentration) can thenbe transformed into an E. coli strain, e.g. TOP10, followed by minipreps and restriction mapping to further verify the plasmid construct.Finally the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome alsotakes advantage of the homologous recombination system in yeast.Typically, a cassette containing a coding region plus control elements(promoter and terminator) and auxotrophic marker is PCR-amplified with ahigh-fidelity DNA polymerase using primers that hybridize to thecassette and contain 40-70 base pairs of sequence homology to theregions 5′ and 3′ of the genomic area where insertion is desired. ThePCR product is then transformed into yeast and plated on mediumcontaining the appropriate compound mixtures that allow selection forthe integrated auxotrophic marker. For example, to integrate “Gene X”into chromosomal location “Y”, the promoter-coding regionX-terminatorconstruct is PCR amplified from a plasmid DNA construct and joined to anautotrophic marker (such as URA3) by either SOE PCR or by commonrestriction digests and cloning. The full cassette, containing thepromoter-coding regionX-terminator-URA3 region, is PCR amplified withprimer sequences that contain 40-70 bp of homology to the regions 5′ and3′ of location “Y” on the yeast chromosome. The PCR product istransformed into yeast and selected on growth media lacking uracil.Transformants can be verified either by colony PCR or by directsequencing of chromosomal DNA.

Expression of a Butanol Biosynthetic Pathway in Lactobacillus plantarum

The Lactobacillus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Bacillus subtilis andStreptococcus can be used for Lactobacillus. Non-limiting examples ofsuitable vectors include pAMβ1 and derivatives thereof (Renault et al.,Gene 183:175-182, 1996); and (O'Sullivan et al., Gene, 137: 227-231,1993); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al., Appl.Environ. Microbiol., 62: 1481-1486, 1996); pMG1, a conjugative plasmid(Tanimoto et al., J. Bacteriol., 184: 5800-5804, 2002); pNZ9520(Kleerebezem et al., Appl. Environ. Microbiol., 63: 4581-4584, 1997);pAM401 (Fujimoto et al., Appl. Environ. Microbiol., 67: 1262-1267,2001); and pAT392 (Arthur et al., Antimicrob. Agents Chemother., 38:1899-1903, 1994). Several plasmids from Lactobacillus plantarum havealso been reported (van Kranenburg R, et al. Appl. Environ. Microbiol.,71: 1223-1230, 2005).

Expression of a Butanol Biosynthetic Pathway in Various EnterococcusSpecies (E. Faecium, E. Gallinarium, and E. Faecalis)

The Enterococcus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Lactobacilli, Bacilliand Streptococci species can be used for Enterococcus species.Non-limiting examples of suitable vectors include pAMβ1 and derivativesthereof (Renault et al., Gene, 183: 175-182, 1996); and (O'Sullivan etal., Gene, 137: 227-231, 1993); pMBB1 and pHW800, a derivative of pMBB1(Wyckoff et al. Appl. Environ. Microbiol., 62: 1481-1486, 1996); pMG1, aconjugative plasmid (Tanimoto et al., J. Bacteriol., 184: 5800-5804,2002); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol., 63:4581-4584, 1997); pAM401 (Fujimoto et al., Appl. Environ. Microbiol.,67: 1262-1267, 2001); and pAT392 (Arthur et al., Antimicrob. AgentsChemother., 38:, 1899-1903, 1994). Expression vectors for E. faecalisusing the nisA gene from Lactococcus can also be used (Eichenbaum etal., Appl. Environ. Microbiol., 64: 2763-2769, 1998). Additionally,vectors for gene replacement in the E. faecium chromosome can be used(Nallaapareddy et al., Appl. Environ. Microbiol., 72: 334-345, 2006).

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates can include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose, maltose, galactose, sucrose, polysaccharides such as starch orcellulose or mixtures thereof and unpurified mixtures from renewablefeedstocks such as cheese whey permeate, cornsteep liquor, sugar beetmolasses, and barley malt. Additionally the carbon substrate can also beone-carbon substrates such as carbon dioxide, or methanol for whichmetabolic conversion into key biochemical intermediates has beendemonstrated. In addition to one and two carbon substratesmethylotrophic microorganisms are also known to utilize a number ofother carbon containing compounds such as methylamine, glucosamine and avariety of amino acids for metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.,[Int. Symp.], 7th (1993), 415-32. (eds): Murrell, J. Collin; Kelly, DonP. Publisher: Intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol., 153: 485-489, 1990). Hence it is contemplated that thesource of carbon utilized in the present invention can encompass a widevariety of carbon containing substrates and will only be limited by thechoice of microorganism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,in some embodiments, the carbon substrates are glucose, fructose, andsucrose, or mixtures of these with C5 sugars such as xylose and/orarabinose for yeasts cells modified to use C5 sugars. Sucrose can bederived from renewable sugar sources such as sugar cane, sugar beets,cassaya, sweet sorghum, and mixtures thereof. Glucose and dextrose canbe derived from renewable grain sources through saccharification ofstarch based feedstocks including grains such as corn, wheat, rye,barley, oats, and mixtures thereof. In addition, fermentable sugars canbe derived from renewable cellulosic or lignocellulosic biomass throughprocesses of pretreatment and saccharification, as described, forexample, in U.S. Patent Appl. Pub. No. 2007/0031918 A1, which is hereinincorporated by reference in its entirety. Biomass refers to anycellulosic or lignocellulosic material and includes materials comprisingcellulose, and optionally further comprising hemicellulose, lignin,starch, oligosaccharides and/or monosaccharides. Biomass can alsocomprise additional components, such as protein and/or lipid. Biomasscan be derived from a single source, or biomass can comprise a mixturederived from more than one source; for example, biomass can comprise amixture of corn cobs and corn stover, or a mixture of grass and leaves.Biomass includes, but is not limited to, bioenergy crops, agriculturalresidues, municipal solid waste, industrial solid waste, sludge frompaper manufacture, yard waste, wood and forestry waste. Examples ofbiomass include, but are not limited to, corn grain, corn cobs, cropresidues such as corn husks, corn stover, grasses, wheat, wheat straw,barley, barley straw, hay, rice straw, switchgrass, waste paper, sugarcane bagasse, sorghum, soy, components obtained from milling of grains,trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers, animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for growth ofthe cultures and promotion of the enzymatic pathway necessary forbutanol production described herein.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C.to about 40° C. in an appropriate medium. Suitable growth media in thepresent invention are common commercially prepared media such as LuriaBertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM)broth or broth that includes yeast nitrogen base, ammonium sulfate, anddextrose (as the carbon/energy source) or YPD Medium, a blend ofpeptone, yeast extract, and dextrose in optimal proportions for growingmost Saccharomyces carevisiae strains. Other defined or synthetic growthmedia can also be used, and the appropriate medium for growth of theparticular microorganism will be known by one skilled in the art ofmicrobiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′,3′-monophosphate (cAMP), can also be incorporated into thefermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred for the initial condition. SuitablepH ranges for the fermentation of yeast are typically between about pH3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 isused for the initial condition. Suitable pH ranges for the fermentationof other microorganisms are between about pH 3.0 to about pH 7.5. In oneembodiment, about pH 4.5 to about pH 6.5 is used for the initialcondition.

Fermentations can be performed under aerobic or anaerobic conditions. Inone embodiment, anaerobic or microaerobic conditions are used forfermentation.

Industrial Batch and Continuous Fermentations

The present processes may employ a batch method of fermentation. Aclassical batch fermentation is a closed system where the composition ofthe medium is set at the beginning of the fermentation and not subjectto artificial alterations during the fermentation. Thus, at thebeginning of the fermentation the medium is inoculated with the desiredmicroorganism or microorganisms, and fermentation is permitted to occurwithout adding anything to the system. Typically, however, a “batch”fermentation is batch with respect to the addition of carbon source andattempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the fermentation isstopped. Within batch cultures cells moderate through a static lag phaseto a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase generally areresponsible for the bulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the medium. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples can be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund(Appl. Biochem. Biotechnol., 36: 227, 1992), herein incorporated byreference.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned medium is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by medium turbidity, is kept constant. Continuous systemsstrive to maintain steady state growth conditions and thus the cell lossdue to the medium being drawn off must be balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing to the rate of product formation are wellknown in the art of industrial microbiology and a variety of methods aredetailed by Brock, supra.

It is contemplated that the present invention can be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells can be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for isobutanol production.

Methods for Butanol Isolation from the Fermentation Medium

Bioproduced butanol can be isolated from the fermentation medium usingmethods known in the art for ABE fermentations (see, e.g., Durre, Appl.Microbial. Biotechnol. 49:639-648 (1998), Groot et al., Process.Biochem. 27:61-75 (1992), and references therein). For example, solidscan be removed from the fermentation medium by centrifugation,filtration, decantation, or the like. Then, the butanol can be isolatedfrom the fermentation medium using methods such as distillation,azeotropic distillation, liquid-liquid extraction, adsorption, gasstripping, membrane evaporation, or pervaporation.

Because butanol forms a low boiling point, azeotropic mixture withwater, distillation can be used to separate the mixture up to itsazeotropic composition. Distillation can be used in combination withanother separation method to obtain separation around the azeotrope.Methods that can be used in combination with distillation to isolate andpurify butanol include, but are not limited to, decantation,liquid-liquid extraction, adsorption, and membrane-based techniques.Additionally, butanol can be isolated using azeotropic distillationusing an entrainer (see, e.g., Doherty and Malone, Conceptual Design ofDistillation Systems, McGraw Hill, N.Y., 2001).

The butanol-water mixture forms a heterogeneous azeotrope so thatdistillation can be used in combination with decantation to isolate andpurify the butanol. In this method, the butanol containing fermentationbroth is distilled to near the azeotropic composition. Then, theazeotropic mixture is condensed, and the butanol is separated from thefermentation medium by decantation. The decanted aqueous phase can bereturned to the first distillation column as reflux. The butanol-richdecanted organic phase can be further purified by distillation in asecond distillation column.

The butanol can also be isolated from the fermentation medium usingliquid-liquid extraction in combination with distillation. In thismethod, the butanol is extracted from the fermentation broth usingliquid-liquid extraction with a suitable solvent. The butanol-containingorganic phase is then distilled to separate the butanol from thesolvent.

Distillation in combination with adsorption can also be used to isolatebutanol from the fermentation medium. In this method, the fermentationbroth containing the butanol is distilled to near the azeotropiccomposition and then the remaining water is removed by use of anadsorbent, such as molecular sieves (Aden et al., LignocellulosicBiomass to Ethanol Process Design and Economics Utilizing Co-CurrentDilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover,Report NREL/TP-510-32438, National Renewable Energy Laboratory, June2002).

Additionally, distillation in combination with pervaporation can be usedto isolate and purify the butanol from the fermentation medium. In thismethod, the fermentation broth containing the butanol is distilled tonear the azeotropic composition, and then the remaining water is removedby pervaporation through a hydrophilic membrane (Guo et al., J. Membr.Sci. 245, 199-210 (2004)).

In situ product removal (ISPR) (also referred to as extractivefermentation) can be used to remove butanol (or other fermentativealcohol) from the fermentation vessel as it is produced, therebyallowing the microorganism to produce butanol at high yields. One methodfor ISPR for removing fermentative alcohol that has been described inthe art is liquid-liquid extraction. In general, with regard to butanolfermentation, for example, the fermentation medium, which includes themicroorganism, is contacted with an organic extractant at a time beforethe butanol concentration reaches a toxic level. The organic extractantand the fermentation medium form a biphasic mixture. The butanolpartitions into the organic extractant phase, decreasing theconcentration in the aqueous phase containing the microorganism, therebylimiting the exposure of the microorganism to the inhibitory butanol.

Liquid-liquid extraction can be performed, for example, according to theprocesses described in U.S. Patent Appl. Pub. No. 2009/0305370, thedisclosure of which is hereby incorporated in its entirety. U.S. PatentAppl. Pub. No. 2009/0305370 describes methods for producing andrecovering butanol from a fermentation broth using liquid-liquidextraction, the methods comprising the step of contacting thefermentation broth with a water immiscible extractant to form atwo-phase mixture comprising an aqueous phase and an organic phase.Typically, the extractant can be an organic extractant selected from thegroup consisting of saturated, mono-unsaturated, poly-unsaturated (andmixtures thereof) C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids,esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, andmixtures thereof. The extractant(s) for ISPR can be non-alcoholextractants. The ISPR extractant can be an exogenous organic extractantsuch as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol,myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid,myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal,lauric aldehyde, 20-methylundecanal, and mixtures thereof.

In some embodiments, the ester can be formed by contacting the alcoholin a fermentation medium with a carboxylic acid (e.g., fatty acids) anda catalyst capable of esterfiying the alcohol with the carboxylic acid,as described in PCT Appn. Pub. No. WO/2011/159998 and U.S. Appl. Pub.No. 20120156738, which are herein incorporated by reference in theirentireties. In such embodiments, the carboxylic acid can serve as anISPR extractant into which the alcohol esters partition. The carboxylicacid can be supplied to the fermentation vessel and/or derived from thebiomass supplying fermentable carbon fed to the fermentation vessel.Lipids present in the feedstock can be catalytically hydrolyzed tocarboxylic acid, and the same catalyst (e.g., enzymes) can esterify thecarboxylic acid with the alcohol. The catalyst can be supplied to thefeedstock prior to fermentation, or can be supplied to the fermentationvessel before or contemporaneously with the supplying of the feedstock.When the catalyst is supplied to the fermentation vessel, alcohol esterscan be obtained by hydrolysis of the lipids into carboxylic acid andsubstantially simultaneous esterification of the carboxylic acid withbutanol present in the fermentation vessel. Carboxylic acid and/ornative oil not derived from the feedstock can also be fed to thefermentation vessel, with the native oil being hydrolyzed intocarboxylic acid. Any carboxylic acid not esterified with the alcohol canserve as part of the ISPR extractant. The extractant containing alcoholesters can be separated from the fermentation medium, and the alcoholcan be recovered from the extractant. The extractant can be recycled tothe fermentation vessel. Thus, in the case of butanol production, forexample, the conversion of the butanol to an ester may reduce the freebutanol concentration in the fermentation medium, shielding themicroorganism from the toxic effect of increasing butanol concentration.In addition, unfractionated grain can be used as feedstock withoutseparation of lipids therein, since the lipids can be catalyticallyhydrolyzed to carboxylic acid, thereby decreasing the rate of build-upof lipids in the ISPR extractant.

In situ product removal can be carried out in a batch mode or acontinuous mode. In a continuous mode of in situ product removal,product is continually removed from the reactor. In a batchwise mode ofin situ product removal, a volume of organic extractant is added to thefermentation vessel and the extractant is not removed during theprocess. For in situ product removal, the organic extractant can contactthe fermentation medium at the start of the fermentation forming abiphasic fermentation medium. Alternatively, the organic extractant cancontact the fermentation medium after the microorganism has achieved adesired amount of growth, which can be determined by measuring theoptical density of the culture. Further, the organic extractant cancontact the fermentation medium at a time at which the product alcohollevel in the fermentation medium reaches a preselected level. In thecase of butanol production according to some embodiments of the presentinvention, the carboxylic acid extractant can contact the fermentationmedium at a time before the butanol concentration reaches a toxic level,so as to esterify the butanol with the carboxylic acid to producebutanol esters and consequently reduce the concentration of butanol inthe fermentation vessel. The ester-containing organic phase can then beremoved from the to fermentation vessel (and separated from thefermentation broth which constitutes the aqueous phase) after a desiredeffective titer of the butanol esters is achieved. In some embodiments,the ester-containing organic phase is separated from the aqueous phaseafter fermentation of the available fermentable sugar in thefermentation vessel is substantially is complete.

EXAMPLES

Construction of Strains

TABLE 8 Strains referenced in the Examples Strain name GenotypeDescription PNY2204 MATa ura3Δ::loxP his3Δ pdc6Δ PCT Publicationpdc1Δ::P[PDC1]-DHAD|ilvD_Sm- No. PDC1t-pUC19-loxP-kanMX-loxP-WO2012033832, P[FBA1]-ALS|alsS_Bs-CYC1t and U.S. Appl.pdc5Δ::P[PDC5]-ADH|sadB_Ax- Pub. No. US PDC5t gpd2Δ::loxP fra2Δ20120237988A1, adh1Δ::UAS(PGK1)P[FBA1]- incorporated kivD_Ll(y)-ADH1therein by reference in their entireties PNY2211 MATa ura3Δ::loxP his3Δpdc6Δ PCT Publication pdc1Δ::P[PDC1]-DHAD|ilvD_Sm- No.PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t WO2012033832,pdc5Δ::P[PDC5]-ADH|sadB_Ax- and U.S. Appl. PDC5t gpd2Δ::loxP fra2Δ Pub.No. US adh1Δ::UAS(PGK1)P[FBA1]- 20120237988A1, kivD_Ll(y)-ADH1tincorporated herein by reference in their entireties PNY2238 MATaura3Δ::loxP his3Δ pdc6Δ herein pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax- PDC5tgpd2Δ::loxP fra2Δ::P[PDC1]- ADH|adh_Hl-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t yprcΔ15Δ::P[PDC5]-ADH|adh_Hl- ADH1t ymr226cΔald6Δ::loxP PNY2259 MATa ura3Δ::loxP his3Δ pdc6Δ hereinpdc1Δ::P[PDC1]-DHAD|ilvD_Sm- PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax- PDC5t gpd2Δ::loxP fra2Δ::P[PDC1]-ADH|adh_Hl-ADH1t adh1Δ::UAS(PGK1)P[FBA1]- kivD_Lg(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_Hl- ADH1t ymr226cΔ ald6Δ::loxP PNY1556 MATaura3Δ::loxP his3Δ pdc6Δ herein pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax- PDC5tgpd2Δ::loxP fra2Δ::P[PDC1]- ADH|adh_Hl-ADH1t adh1Δ::UAS(PGK1)P[FBA1]-kivD_Lg(y)-ADH1t yprcΔ15Δ::P[PDC5]-ADH|adh_Hl- ADH1tConstruction of PNY1528 (hADH Integrations in PNY2211)

Deletions/integrations were created by homologous recombination with PCRproducts containing regions of homology upstream and downstream of thetarget region and the URA3 gene for selection of transformants. The URA3gene was removed by homologous recombination to create a scarlessdeletion/integration.

YPRCΔ15 Deletion and Horse Liver adh Integration

The YPRCΔ15 locus was deleted and replaced with the horse liver adhgene, codon optimized for expression in Saccharomyces cerevisiae, alongwith the PDC5 promoter region (538 bp) from Saccharomyces cerevisiae andthe ADH1 terminator region (316 bp) from Saccharomyces cerevisiae. Thescarless cassette for the YPRCΔ15 deletion-P[PDC5]-adh_HL(y)-ADH1tintegration was first cloned into plasmid pUC19-URA3MCS.

Fragments A-B-U-C were amplified using Phusion High Fidelity PCR MasterMix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNAas template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen;Valencia, Calif.). YPRCA15 Fragment A was amplified from genomic DNAwith primer oBP622 (SEQ ID NO: 269), containing a KpnI restriction site,and primer oBP623 (SEQ ID NO: 270), containing a 5′ tail with homologyto the 5′ end of YPRCΔ15 Fragment B. YPRCΔ15 Fragment B was amplifiedfrom genomic DNA with primer oBP624 (SEQ ID NO: 271), containing a 5′tail with homology to the 3′ end of YPRCΔ15 Fragment A, and primeroBP625 (SEQ ID NO: 272), containing a FseI restriction site. PCRproducts were purified with a PCR Purification kit (Qiagen). YPRCΔ15Fragment A-YPRCΔ15 Fragment B was created by overlapping PCR by mixingthe YPRCΔ15 Fragment A and YPRCΔ15 Fragment B PCR products andamplifying with primers oBPS22 (SEQ ID NO: 269) and oBP625 (SEQ ID NO:272). The resulting PCR product was digested with KpnI and FseI andligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCSafter digestion with the appropriate enzymes. YPRCΔ15 Fragment C wasamplified from genomic DNA with primer oBP626 (SEQ ID NO: 273),containing a NofI restriction site, and primer oBP627 (SEQ ID NO: 274),containing a PacI restriction site. The YPRCΔ15 Fragment C PCR productwas digested with NotI and PacI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing YPRCΔ15 Fragments AB. ThePDC5 promoter region was amplified from CEN.PK 113-7D genomic DNA withprimer HY21 (SEQ ID NO: 275), containing an AscI restriction site, andprimer HY24 (SEQ ID NO: 276), containing a 5′ tail with homology to the5′ end of adh_HI(y). adh_HI(y)-ADH1t was amplified from pBP915 (SEQ IDNO: 279) with primers HY25 (SEQ ID NO: 277), containing a 5′ tail withhomology to the 3′ end of P[PDC5], and HY4 (SEQ ID NO: 278), containinga PmeI restriction site. PCR products were purified with a PCRPurification kit (Qiagen). P[PDC5]-adh_HL(y)-ADH1t was created byoverlapping PCR by mixing the P[PDC5] and adh_HL(y)-ADH1t PCR productsand amplifying with primers HY21 (SEQ ID NO: 275) and HY4 (SEQ ID NO:278).The resulting PCR product was digested with AscI and PmeI andligated with T4 DNA ligase into the corresponding sites of the plasmidcontaining YPRCΔ15 Fragments ABC. The entire integration cassette wasamplified from the resulting plasmid with primers oBP622 (SEQ ID NO:269) and oBP627 (SEQ ID NO: 274).

Competent cells of PNY2211 were made and transformed with the YPRCΔ15deletion-P[PDC5]-adh_HL(y)-ADH1t integration cassette PCR product usinga Frozen-EZ Yeast Transformation II kit (Zymo Research; Orange, Calif.).Transformation mixtures were plated on synthetic complete media lackinguracil supplemented with 1% ethanol at 30° C. Transformants werescreened for by PCR with primers URA3-end F (SEQ ID NO: 280) and oBP637(SEQ ID NO: 283). Correct transformants were grown in YPE (1% ethanol)and plated on synthetic complete medium supplemented with 1% EtOH andcontaining 5-fluoro-orotic acid (0.1%) at 30 C to select for isolatesthat lost the URA3 marker. The deletion of YPRCΔ15 and integration ofP[PDC5]-adh_HL(y)-ADH1t were confirmed by PCR with external primersoBP636 (SEQ ID NO: 281) and oBP637 (SEQ ID NO: 282) using genomic DNAprepared with a YeaStar Genomic DNA kit (Zymo Research). A correctisolate of the following genotype was selected for further modification:CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_LI(y)-ADH1t yprcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t.

Horse Liver Adh Integration at Fra2Δ

The horse liver adh gene, codon optimized for expression inSaccharomyces cerevisiae, along with the PDC1 promoter region (870 bp)from Saccharomyces cerevisiae and the ADH1 terminator region (316 bp)from Saccharomyces cerevisiae, was integrated into the site of the fra2deletion. The scarless cassette for the fra2Δ-P[PDC1]-adh_HL(y)-ADH1tintegration was first cloned into plasmid pUC19-URA3MCS.

Fragments A-B-U-C were amplified using Phusion High Fidelity PCR MasterMix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNAas template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen;Valencia, Calif.). fra2Δ Fragment C was amplified from genomic DNA withprimer oBP695 (SEQ ID NO: 287), containing a NotI restriction site, andprimer oBP696 (SEQ ID NO: 288), containing a PacI restriction site. Thefra2Δ Fragment C PCR product was digested with NotI and PacI and ligatedwith T4 DNA ligase into the corresponding sites of pUC19-URA3MCS. fra2ΔFragment B was amplified from genomic DNA with primer oBP693 (SEQ ID NO:285), containing a PmeI restriction site, and primer oBP694 (SEQ ID NO:286), containing a FseI restriction site. The resulting PCR product wasdigested with PmeI and FseI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing fra2Δ fragment C afterdigestion with the appropriate enzymes. fra2Δ Fragment A was amplifiedfrom genomic DNA with primer oBP691 (SEQ ID NO: 283), containing BamHIand AsiSI restriction sites, and primer oBP692 (SEQ ID NO: 284),containing AscI and Swal restriction sites. The fra2Δ fragment A PCRproduct was digested with BamHI and AscI and ligated with T4 DNA ligaseinto the corresponding sites of the plasmid containing fra2Δ fragmentsBC after digestion with the appropriate enzymes. The PDC1 promoterregion was amplified from CEN.PK 113-7D genomic DNA with primer HY16(SEQ ID NO: 289), containing an AscI restriction site, and primer HY19(SEQ ID NO: 290), containing a 5′ tail with homology to the 5′ end ofadh_HI(y). adh_HI(y)-ADH1t was amplified from pBP915 with primers HY20(SEQ ID NO: 291), containing a 5′ tail with homology to the 3′ end ofP[PDC1], and HY4 (SEQ ID NO: 278), containing a PmeI restriction site.PCR products were purified with a PCR Purification kit (Qiagen).P[PDC1]-adh_HL(y)-ADH1t was created by overlapping PCR by mixing theP[PDC1] and adh_HL(y)-ADH1t PCR products and amplifying with primersHY16 (SEQ ID NO: 289) and HY4 (SEQ ID NO: 278).The resulting PCR productwas digested with AscI and PmeI and ligated with T4 DNA ligase into thecorresponding sites of the plasmid containing fra2Δ Fragments ABC. Theentire integration cassette was amplified from the resulting plasmidwith primers oBP691 (SEQ ID NO: 283) and oBP696 (SEQ ID NO: 288).

Competent cells of the PNY2211 variant with adh_HI(y) integrated atYPRCΔ15 were made and transformed with the fra2Δ-P[PDC1]-adh_HL(y)-ADH1tintegration cassette PCR product using a Frozen-EZ Yeast TransformationII kit (Zymo Research). Transformation mixtures were plated on syntheticcomplete media lacking uracil supplemented with 1% ethanol at 30° C.Transformants were screened for by PCR with primers URA3-end F (SEQ IDNO: 280) and oBP731 (SEQ ID NO: 293). Correct transformants were grownin YPE (1% ethanol) and plated on synthetic complete medium supplementedwith 1% EtOH and containing 5-fluoro-orotic acid (0.1%) at 30° C. toselect for isolates that lost the URA3 marker. The integration ofP[PDC1]-adh_HL(y)-ADH1t was confirmed by colony PCR with internal primerHY31 (SEQ ID NO: 294) and external primer oBP731 (SEQ ID NO: 293) andPCR with external primers oBP730 (SEQ ID NO: 292) and oBP731 (SEQ ID NO:293) using genomic DNA prepared with a YeaStar Genomic DNA kit (ZymoResearch). A correct isolate of the following genotype was designatedPNY1528: CEN.PK 113-7D MATa ura3Δ:loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δ::P[PDC1]-ADH|adh_HI-ADH1tadh1Δ::UAS(PGK1)P[FBA1]-kivD_LI(y)-ADH1typrcΔ15Δ::P[PDC5]-ADH|adh_HI-ADH1t.

PNY2237 (Scarless YMR226c Deletion)

The gene YMR226c was deleted from S. cerevisiae strain PNY1528 byhomologous recombination using a PCR amplified 2.0 kb linear scarlessdeletion cassette. The cassette was constructed from spliced PCRamplified fragments comprised of the URA3 gene, along with its nativepromoter and terminator as a selectable marker, upstream and downstreamhomology sequences flanking the YMR226c gene chromosomal locus topromote integration of the deletion cassette and removal of the nativeintervening sequence and a repeat sequence to promote recombination andremoval of the URA3 marker. Forward and reverse PCR primers (N1251 andN1252, SEQ ID NOs: 295 and 296, respectively), amplified a 1,208 bp URA3expression cassette originating from pLA33 (pUC19::loxP-URA3-loxP (SEQID NO: 303)). Forward and reverse primers (N1253 and N1254, SEQ ID NOs:297 and 298, respectively), amplified a 250 bp downstream homologysequence with a 3′ URA3 overlap sequence tag from a genomic DNApreparation of S. cerevisiae strain PNY2211 (above). Forward and reversePCR primers (N1255 and N1256, SEQ ID NOs: 299 and 300, respectively)amplified a 250 bp repeat sequence with a 5′ URA3 overlap sequence tagfrom a genomic DNA preparation of S. cerevisiae strain PNY2211. Forwardand reverse PCR primers (N1257 and N1258, SEQ ID NOs: 301 and 302,respectively) amplified a 250 bp upstream homology sequence with a 5′repeat overlap sequence tag from a genomic DNA preparation of S.cerevisiae strain PNY2211.

Approximately 1.5 μg of the PCR amplified cassette was transformed intostrain PNY1528 (above) made competent using the ZYMO Research FrozenYeast Transformation Kit and the transformation mix plated on SE1.0%-uracil and incubated at 30° C. for selection of cells with anintegrated ymr226cΔ::URA3 cassette. Transformants appearing after 72 to96 hours are subsequently short-streaked on the same medium andincubated at 30° C. for 24 to 48 hours. The short-streaks are screenedfor ymr226cΔ::URA3 by PCR, with a 5′ outward facing URA3 deletioncassette-specific internal primer (N1249, SEQ ID NO: 312) paired with aflanking inward facing chromosome-specific primer (N1239, SEQ ID NO:310) and a 3′ outward-facing URA3 deletion cassette-specific primer(N1250, SEQ ID NO: 313) paired with a flanking inward-facingchromosome-specific primer (N1242, SEQ ID NO: 311). A positive PNY1528ymr226cΔ::URA3 PCR screen resulted in 5′ and 3′ PCR products of 598 and726 bp, respectively.

Three positive PNY1528 ymr226cΔ::URA3 clones were picked and culturedovernight in a YPE 1% medium of which 100 μL was plated on YPE 1%+5-FOAfor marker removal. Colonies appearing after 24 to 48 hours were PCRscreened for marker loss with 5′ and 3′ chromosome-specific primers(N1239 and N1242). A positive PNY1528 ymr226cΔ markerless PCR screenresulted in a PCR product of 801 bp. Multiple clones were obtained andone was designated PNY2237.

PNY2238 and PNY2243 (ALD6 Deletion Strains)

A vector was designed to replace the ALD6 coding sequence with a Cre-loxrecyclable URA3 selection marker. Sequences 5′ and 3′ of ALD6 wereamplified by PCR (primer pairs N1179 and N1180 and N1181 and N1182,respectively; SEQ ID NOs: 304, 305, 306, and 307, respectively). Aftercloning these fragments into TOPO vectors (Invitrogen Cat. No.K2875-J10) and sequencing (M13 forward (SEQ ID NO:314) and reverse (SEQID NO:315) primers), the 5′ and 3′ flanks were cloned into pLA33(pUC19::loxP::URA3::loxP) (SEQ ID NO:303) at the EcoRI and SphI sites,respectively. Each ligation reaction was transformed into E. coli Stbl3cells, which were incubated on LB Amp plates to select fortransformants. Proper insertion of sequences was confirmed by PCR(primers M13 forward (SEQ ID NO: 314) and N1180 (SEQ ID NO:306) and M13reverse (SEQ ID NO:315) and N1181 (SEQ ID NO:306, respectively).

The vector described above (pUC19::ald6Δ::loxP-URA3-loxP) was linearizedwith AhdI and transformed into PNY1528 and PNY2237 using the standardlithium acetate method (except that incubation of cells with DNA wasextended to 2.5 h). Transformants were obtained by plating on syntheticcomplete medium minus uracil that provided 1% ethanol as the carbonsource. Patched transformants were screened by PCR to confirm thedeletion/integration, using primers N1212 (SEQ ID NO: 308) and N1180 (5′end) (SEQ ID NO: 305) and N1181 (SEQ ID NO: 306) and N1213 (SEQ ID NO:309) (3′ end). A plasmid carrying Cre recombinase (pRS423::GAL1p-Cre=SEQID No. 324) was transformed into the strain using histidine markerselection. Transformants were passaged on YPE supplemented with 0.5%galactose. Colonies were screened for resistance to 5-FOA (loss of URA3marker) and for histidine auxotrophy (loss of the Cre plasmid). Properremoval of the URA3 gene via the flanking loxP sites was confirmed byPCR (primers N1262 and N1263, SEQ ID NOs: 316 and 317, respectively).Additionally, primers internal to the ALD6 gene (N1230 and N1231; SEQ IDNOs: 322 and 323, respectively) were used to insure that no merodiploidswere present. Finally, ald6Δ::loxP clones were screened by PCR toconfirm that a translocation between ura3Δ:loxP (N1228 and N1229, SEQ IDNOs: 320 and 321) and gpd2Δ::loxP (N1223 and N1225, SEQ ID NOs: 318 and319) had not occurred. Two positive clones were identified fromscreening of transformants of PNY1528. Clone B has been designatedPNY2243. Three positive clones were identified from screeningtransformants of PNY2237. Clones E and K were both assessed forisobutanol production at small scale (below). Although statisticallyidentical in most parameters, Clone E was selected (PNY2238) for furtherdevelopment.

Construction of Strain PNY2259

The purpose of this example is to describe the assembly of theconstructs used to replace the chromosomal copy of kivD_LI(y) in PNY2238at the adh1Δ locus with kivD_Lg(y).

The deletion/integration was created by homologous recombination withPCR products containing regions of homology upstream and downstream ofthe target region and the URA3 gene for selection of transformants. TheURA3 gene was removed by homologous recombination to create a scarlessdeletion/integration. The plasmid to integrate kivD_Lg(y) was derivedfrom a plasmid constructed to integrate UAS(PGK1)P[FBA1]-kivD_LI(y) intothe ADH1 locus of Saccharomyces cerevisiae. Construction of the plasmidused to integrate UAS(PGK1)P[FBA1]-kivD_LI(y) into the ADH1 locus isdescribed below. The plasmids were constructed in pUC19-URA3MCS.

Construction of the ADH1 Deletion/UAS(PGK1)P[FBA1]-kivD_LI(y),Integration Plasmid

The kivD coding region from Lactococcus lactis codon optimized forexpression in Saccharomyces cerevisiae, kivD_LI(y), was amplified usingpLH468 (SEQ ID NO: 335) as template with primer oBP562 (SEQ ID NO: 325),containing a PmeI restriction site, and primer oBP563 (SEQ ID NO: 326),containing a 5′ tail with homology to the 5′ end of ADH1 Fragment B.ADH1 Fragment B was amplified from Saccharomyces cerevisiae CEN.PK113-7D genomic DNA with primer oBP564 (SEQ ID NO: 327), containing a 5′tail with homology to the 3′ end of kivD_LI(y), and primer oBP565 (SEQID NO: 328), containing a FseI restriction site. PCR products werepurified with a PCR Purification kit (Qiagen; Valencia, Calif.).kivD_LI(y)-ADH1 Fragment B was created by overlapping PCR by mixing thekivD_LI(y) and ADH1 Fragment B PCR products and amplifying with primersoBP562 (SEQ ID NO: 325) and oBP565 (SEQ ID NO: 328), The resulting PCRproduct was digested with PmeI and FseI and ligated with T4 DNA ligaseinto the corresponding sites of pUC19-URA3MCS after digestion with theappropriate enzymes. ADH1 Fragment A was amplified from genomic DNA withprimer oBP505 (SEQ ID NO: 329), containing a SacI restriction site, andprimer oBP506 (SEQ ID NO: 330), containing an AscI restriction site. TheADH1 Fragment A PCR product was digested with SacI and AscI and ligatedwith T4 DNA ligase into the corresponding sites of the plasmidcontaining kivD_LI(y)-ADH1 Fragment B. ADH1 Fragment C was amplifiedfrom genomic DNA with primer oBP507 (SEQ ID NO: 331), containing a PacIrestriction site, and primer oBP508 (SEQ ID NO: 332), containing a SaIIrestriction site. The ADH1 Fragment C PCR product was digested with PacIand SaII and ligated with T4 DNA ligase into the corresponding sites ofthe plasmid containing ADH1 Fragment A-kivD_LI(y)-ADH1 Fragment B. Thehybrid promoter UAS(PGK1)-P_(FBA1) (SEQ ID NO: 264) was amplified fromvector pRS316-UAS(PGK1)-P_(FBA1)-GUS with primer oBP674 (SEQ ID NO:333), containing an AscI restriction site, and primer oBP675 (SEQ ID NO:334), containing a PmeI restriction site. The UAS(PGK1)-P_(FBA1) PCRproduct was digested with AscI and PmeI and ligated with T4 DNA ligaseinto the corresponding sites of the plasmid containing kivD_LI(y)-ADH1Fragments ABC to generate pBP1181.

Construction of pBP1716 and pBP1719

kivD_LI(y) was removed from the ADH1deletion/UAS(PGK1)P[FBA1]-kivD_LI(y) integration plasmid pBP1181. Theplasmid was digested with Pmel and FseI and the large DNA fragment waspurified on an agarose gel followed by a gel extraction kit (Qiagen).ADH1 fragment B was amplified from pBP1181 with primer oBP821 (SEQ IDNO: 336), containing a PmeI restriction site, and primer oBP484 (SEQ IDNO: 337), containing a FseI restriction site. The ADH1 fragment B PCRproduct was digested with PmeI and FseI and ligated with T4 DNA ligaseinto the corresponding sites of the gel purified large DNA fragment. APCR fragment corresponding to the 3′ 500 bp of kivD_LI(y) was clonedinto the resulting vector for the targeted deletion of kivD_LI(y) inPNY1528. The fragment was amplified from pBP1181 with primers oBP822(SEQ ID NO: 338), containing a NotI restriction site, and oBP823 (SEQ IDNO: 339), containing a Pad restriction site. The fragment was digestedwith NotI and PacI and ligated with T4 DNA ligase into the correspondingsites downstream of URA3 in the above plasmid with the kivD_LI(y)deletion after digestion with the appropriate restriction enzymes. Theresulting plasmid was designated pBP1716.

The kivD coding region from Listeria grayi codon optimized forexpression in Saccharomyces cerevisiae (SEQ ID NO: 340), kivD_Lg(y), wassynthesized by DNA2.0 (Menlo Park, Calif.). kivD_Lg(y) was amplifiedwith primers oBP828 (SEQ ID NO: 341), containing a PmeI restrictionsite, and oBP829 (SEQ ID NO: 342) containing a PmeI restriction site.The resulting PCR product was digested with PmeI and ligated with T4 DNAligase into the corresponding site in pBP1716 after digestion with theappropriate enzyme. The orientation of the cloned gene was checked byPCR with primers FBAp-F (SEQ ID NO: 343) and oBP829 (SEQ ID NO: 342). Anisolate with kivD_Lg(y) in the correct orientation was designatedpBP1719.

Construction of Strain PNY2259

The kivD_μg(y) deletion/kivD_Lg(y) integration cassette was amplifiedfrom pBP1719 with primers oBP505 (SEQ ID NO: 329) and oBP823 (SEQ ID NO:339). Competent cells of the PNY2238 were made and transformed with thePCR product using a Frozen-EZ Yeast Transformation II kit (ZymoResearch; Orange, Calif.). Transformation mixtures were plated onsynthetic complete media lacking uracil supplemented with 1% ethanol at30C. Transformant strains were screened by PCR (JumpStart™ REDTaq (c)ReadyMix™) using primers Ura3-end F (SEQ ID NO: 280) and HY-50 (SEQ IDNO: 344). Transformants were grown in YPE (1% ethanol) and plated onsynthetic complete medium supplemented with 1% EtOH and containing5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost theURA3 marker. The deletion of kivD_LI(y) and integration of kivD_Lg(y)was confirmed by PCR with primers HY-50 and oBP834 (SEQ ID NO: 345). Onecorrect isolate contained kivD_Lg(y) at the same locus and expressedfrom the same promoter as kivD_LI(y) in PNY2238 was designated PNY2259.

Construction of Strain PNY1556

Described here is the assembly of the constructs used to replace thechromosomal copy of kivD_LI(y) in PNY1528 at the adh1Δ locus withkivD_Lg(y) and construction of strain PNY1556 expressing the kivD_Lg(y)gene. Deletions/integrations were created by homologous recombinationwith PCR products containing regions of homology upstream and downstreamof the target region and the URA3 gene for selection of transformants asdescribed in the previous section. The plasmid to integrate kivD_Lg(y)was derived from a plasmid constructed to integrateUAS(PGK1)P[FBA1]-kivD_LI(y) into the ADH1 locus of Saccaromycescerevisiae. Construction of the plasmid used to integrateUAS(PGK1)P[FBA1]-kivD_LI(y) into the ADH1 locus is described below. Theplasmids were constructed in pUC19-URA3MCS.

Construction of the ADH1 deletion/UAS(PGK1)P[FBA1]-kivD_LI(y)Integration Plasmid

The kivD coding region from Lactococcus lactis codon optimized forexpression in Saccharomyces cerevisiae, kivD_LI(y), was amplified usingpLH468 (SEQ ID NO: 129) as template with primer oBP562 (SEQ ID NO: 384),containing a PmeI restriction site, and primer oBP563 (SEQ ID NO: 385),containing a 5′ tail with homology to the 5′ end of ADH1 Fragment B.ADH1 Fragment B was amplified from Saccharomyces cerevisiae CEN.PK113-7D genomic DNA with primer oBP564 (SEQ ID NO: 386), containing a 5′tail with homology to the 3′ end of kivD Ll(y), and primer oBP565 (SEQID NO: 387), containing a FseI restriction site. PCR products werepurified with a PCR Purification kit (Qiagen; Valencia, Calif.).kivD_LI(y)-ADH1 Fragment B was created by overlapping PCR by mixing thekivD_LI(y) and ADH1 Fragment B PCR products and amplifying with primersoBP562 (SEQ ID NO: 384) and oBP565 (SEQ ID NO 387). The resulting PCRproduct was digested with PmeI and FseI and ligated with T4 DNA ligaseinto the corresponding sites of pUC19-URA3MCS after digestion with theappropriate enzymes. ADH1 Fragment A was amplified from genomic DNA withprimer oBP505 (SEQ ID NO: 329), containing a SacI restriction site, andprimer oBP506 (SEQ ID NO: 330), containing an AscI restriction site. TheADH1 Fragment A PCR product was digested with SacI and AscI and ligatedwith T4 DNA ligase into the corresponding sites of the plasmidcontaining kivD_LI(y)-ADH1 Fragment B. ADH1 Fragment C was amplifiedfrom genomic DNA with primer oBP507 (SEQ ID NO: 331), containing a JoPad restriction site, and primer oBP508 (SEQ ID NO: 332), containing aSaII restriction site. The ADH1 Fragment C PCR product was digested withPad and SaII and ligated with T4 DNA ligase into the corresponding sitesof the plasmid containing ADH1 Fragment A-kivD_LI(y)-ADH1 Fragment B.The hybrid promoter UAS(PGK1)-P_(FBA1) was amplified from vectorpRS316-UAS(PGK1)-PFBA1-GUS (SEQ ID NO: 389) with primer oBP674 (SEQ IDNO: 333), containing an AscI restriction site, and primer oBP675 (SEQ IDNO: 334), containing a PmeI restriction site. The UAS(PGK1)-P_(FBA1) PCRproduct was digested with AscI and PmeI and ligated with T4 DNA ligaseinto the corresponding sites of the plasmid containing kivD_LI(y)-ADH1Fragments ABC to generate pBP1181.

kivD_LI(y) was removed from the ADH1deletion/UAS(PGK1)P[FBA1]-kivD_LI(y) integration plasmid pBP1181. Theplasmid was digested with PmeI and FseI and the large DNA fragment waspurified on an agarose gel followed by a gel extraction kit (Qiagen).ADH1 fragment B was amplified from pBP1181 with primer oBP821 (SEQ IDNO: 336), containing a PmeI restriction site, and primer oBP484 (SEQ IDNO: 337), containing a FseI restriction site. The ADH1 fragment B PCRproduct was digested with PmeI and FseI and ligated with T4 DNA ligaseinto the corresponding sites of the gel purified large DNA fragment. APCR fragment corresponding to the 3′ 500 bp of kivD_LI(y) was clonedinto the resulting vector for the targeted deletion of kivD_LI(y) inPNY1528. The fragment was amplified from pBP1181 with primers oBP822(SEQ ID NO: 338), containing a NotI restriction site, and oBP823 (SEQ IDNO: 339), containing a PacI restriction site. The fragment was digestedwith NotI and PacI and ligated with T4 DNA ligase into the correspondingsites downstream of URA3 in the above plasmid with the kivD_LI(y)deletion after digestion with the appropriate restriction enzymes. Theresulting plasmid was designated pBP1716.

The kivD coding region from Listeria grayi codon optimized forexpression in Saccharomyces cerevisiae (SEQ ID NO: 390), kivD_Lg(y), wassynthesized by DNA2.0 (Menlo Park, Calif.). kivD_Lg(y) was amplifiedwith primers oBP828 (SEQ ID NO: 341), containing a PmeI restrictionsite, and oBP829 (SEQ ID NO: 342) containing a PmeI restriction site.The resulting PCR product was digested with PmeI and ligated with T4 DNAligase into the corresponding site in pBP1716 after digestion with theappropriate enzyme. The orientation of the cloned gene was checked byPCR with primers FBAp-F (SEQ ID NO: 343) and oBP829 (SEQ ID NO: 342). Anisolate with kivD_Lg(y) in the correct orientation was designatedpBP1719.

The kivD_LI(y) deletion/kivD_Lg(y) integration cassette was amplifiedfrom pBP1719 with primers oBP505 (SEQ ID NO: 329) and oBP823 (SEQ ID NO:339). Competent cells of the PNY1528 were made and transformed with thePCR product using a Frozen-EZ Yeast Transformation II kit (ZymoResearch; Orange, Calif.). Transformation mixtures were plated onsynthetic complete media lacking uracil supplemented with 1% ethanol at30C. Transformants were grown in YPE (1% ethanol) and plated onsynthetic complete medium supplemented with 1% EtOH and containing5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost theURA3 marker. The deletion of kivD_LI(y) and integration of kivD_Lg(y)was confirmed by PCR with primers oBP674 (SEQ ID NO: 333) and oBP830(SEQ ID NO: 391) using genomic DNA prepared with a YeaStar Genomic DNAkit (Zymo Research). A correct isolate contained kivD_Lg(y) at the samelocus and expressed from the same promoter as kivD_LI(y) in PNY1528 andwas designated PNY1556.

General Methods for Examples 1-4

Yeast Transformation with Plasmids:

Frozen aliquots of competent cells of yeast strains to be transformedwith KARI-containing plasmids were made as follows. For cases where theKARI and DHAD genes were on separate plasmids, competent cells werefirst made from cells harboring the DHAD-containing plasmid (e.g.pBP915). Those competent cells were then transformed with theKARI-containing plasmids.

10 mL of growth medium (YPE for plasmid-free yeast, SE -His for yeastharboring a DHAD-containing plasmid such as pBP915) was inoculated witha loop of cells from a freshly patched plate. The culture was shakenovernight at 30° C. The OD_(600 nm) of the overnight culture wasmeasured with a cuvette spectrophotometer and a calculated amount ofcells was used to inoculate a subsequent 30 mL culture at an initialOD_(600 nm) of 0.2. The growth medium was YPE for plasmid-free yeast orSE -His for yeast harboring a DHAD-containing plasmid such as pBP915.The flask containing the 30 mL culture was shaken for approximately 8hours at 30° C. until the OD_(600 nm) reached approximately 0.7 to 1.0.25 mL of the culture was centrifuged at 4000 rpm for 10 minutes in aswinging-bucket rotor. The supernatant was discarded and the pellet wasresuspended in 10 mL of EZ 1 solution (from the Frozen-EZ YeastTransformation II Kit, Catalog #T2001, Zymo Research, Orange, Calif.).The cells were recentrifuged as above and the supernatant was discarded.The pellet was resuspended 1.75 mL EZ 2 solution (Zymo Research) andaliquoted into microfuge tubes for freezing. The aliquots of competentcells were placed inside two concentric styrofoam boxes to prolong thefreezing process. The assembly was placed in a freezer at −80° C. Thealiquots were transferred out of the concentric styrofoam boxes intostandard freezer boxes after no less than 12 hours.

Yeast cells were transformed with plasmids as follows: For each plasmidto be transformed, 8 μL (300-400 ng) of plasmid DNA solution wasdispensed into a 1.5 mL microfuge tube. Aliquots of frozen competentyeast cells were thawed on ice. 20 μL of thawed competent yeast cellswas added to each tube and mixed with the DNA by pipette. 200 μL of EZ 3Solution (Zymo Research) was added to each tube and mixed by pipetting.The mixtures were incubated at 30° C. for approximately 2 hours withintermittent vortex mixing every 30-45 minutes. 150 μL of eachtransformation mixture was spread onto SE -Ura or SE -Ura -His plates(as appropriate to the plasmid(s) in the experiment). The plates weresealed in zip-top plastic bags and incubated at 30° C. until coloniesappeared.

Yeast Cultivation Conditions

Aerobic cultivation medium: SE -Ura medium (or SE -Ura -His for2-plasmid system experiments) with 2 g/l ethanol.

Anaerobic cultivation medium: SEG -Ura (or SEG -Ura -His for 2-plasmidsystem experiments) with 30 g/l glucose and 1 g/l ethanol, supplementedwith 10 mg/l ergosterol, 50 mM MES buffer (pH 5.5), 30 mg/l thiamine,and 30 mg/l nicotinic acid.

48-well plates: Axygen catalog #P-5ML-48-C—S, 5 ml/well total volume,culture volume of 1.5 ml/well.

Plates were covered with a permeable adhesive film for aerobiccultivation. Plates were shaken at 225 rpm at 30° C. For anaerobiccultivation, freshly inoculated plates covered with permeable film werepurged of oxygen by equilibration in an anaerobic chamber for 2 hours.The plate covers were then exchanged for adhesive aluminum covers andeach plate was placed into an airtight plastic box along with a freshoxygen scavenger pack. The entire assembly (plate(s) and oxygenscavenger pack inside a sealed airtight plastic box) was removed fromthe anaerobic chamber and shaken at 225 rpm at 30° C.

Experimental Protocol

Single yeast colonies on SE -Ura or SE -Ura -His agar plates (asappropriate for the plasmid(s) in the strains used) were streaked ontofresh agar plates of the same type and incubated at 30° C. until densepatches of cells had grown. Liquid precultures in 48-well plates wereinoculated with loops of these cells for initial aerobic cultivation.After shaking overnight, the OD600 of each culture well was measured bytransferring 0.15 ml of each well into a flat-bottom 96-well plate andmeasuring the absorbance of each well at 600 nm with a Molecular Devicesplate reader. A linear transformation based on anexperimentally-determined calibration line was applied to these platereader-measured optical densities to convert them into comparableabsorbance values for a cuvette-based spectrophotometer.

A calculated portion of each aerobic preculture well was inoculated intothe corresponding well of a fresh 48-well plate with 1.5 ml of theanaerobic cultivation medium, to achieve an initial OD600 (in cuvettespectrophotometer absorbance units) of 0.2. In the process ofinoculating the fresh plate, the aerobic preculture plate wascentrifuged, the supernatant was removed from each well, and the cellsin each well were resuspended in fresh anaerobic cultivation medium, inorder to minimize carryover of metabolites from one cultivation to thenext. This anaerobic cultivation plate was shaken for 2-3 days,depending on the experiment. The isobutanol concentration in the culturesupernatants was measured by HPLC (either with a mass spectrometricdetector or a refractive-index detector).

A follow-up anaerobic cultivation (“Passaging Cycle #2”) was initiatedfrom the first anaerobic cultivation as follows: A calculated portion ofeach anaerobic culture well was inoculated into the corresponding wellof a fresh 48-well plate with 1.5 ml of the anaerobic cultivationmedium, to achieve an initial OD600 (in cuvette spectrophotometer units)of 0.2. In the process of inoculating the fresh plate, the growth platewas centrifuged, the supernatant was removed from each well, and thecells in each well were resuspended in fresh anaerobic cultivationmedium, in order to minimize carryover of metabolites from one passagingcycle to the next. The follow-up (second-cycle) anaerobic cultivationplate was shaken for 2-3 days, depending on the experiment. Theisobutanol concentration in the culture supernatants was measured byHPLC as above.

Strain and Growth Medium Details were as follows for Examples 1-5:

Example 1: PNY2204 transformed with two plasmids: 1) a KARI variantplasmid based on SEQ ID NO: 162 and 2) the plasmid given as SEQ ID NO:163 for expression of S. mutans DHAD and growth media lacking uracil andhistidine.

Example 2: PNY2238 transformed with two plasmids (SEQ ID NO: 163 and aKARI-variant plasmid based on SEQ ID NO: 162), growth media lackinguracil and histidine.

Example 3: PNY2238 transformed with two plasmids (SEQ ID NO: 163 and aKARI-variant plasmid based on SEQ ID NO: 162 as in Example 1), growthmedia lacking uracil and histidine.

Example 4: PNY2259 transformed with a single plasmid, growth medialacking uracil.

Example 5: PNY1556 transformed with a single plasmid, growth medialacking uracil.

To generate the KARI-variant plasmid for strains transformed with twoplasmids (above), coding sequences for KARI variants were generated byDNA 2.0 (Menlo Park, Calif.) and were subcloned into a modified vectorbased on SEQ ID NO: 162. An example resultant KARI-variant plasmid(containing the coding sequence for KARI variant 65139; FIG. 7) is givenas SEQ ID NO: 347. Such KARI variant plasmids were used in combinationwith the plasmid given as SEQ ID NO: 163 as described above.

To generate the single plasmid, coding sequences for KARI variants weregenerated by DNA 2.0 (Menlo Park, Calif.) and were subcloned into amodified “single-plasmid vector” (SEQ ID NO: 162, FIG. 3; modifiedplasmid sequence given as SEQ ID NO: 348), replacing the K9D3 KARIcoding region. An example of a resulting plasmid (for expression of KARIvariant SEQ ID NO: 159, “76437” and also known as “R9E8”) is given asSEQ ID NO: 349, FIG. 8.

Example 1 Variants Generated and Tested in an Isobutanol ProductionPathway

TABLE 9 KARI Variants SEQ ID Variant Amino Acid Substitutions NO:Identifier (relative to JEA1) 3 54654 P47F S292A S322A 4 54655 I13LY286F I291V 5 54656 P47F V76I A336R 6 54657 Y286F I291V A336R 7 54658P47F I291V A336G 8 54659 L88V Y286F A336R 9 54660 V76I I152V A336G 1054661 I13L V53A I291V 11 54662 I152V Y286F S322A 12 54663 V76I A268ES292A 13 54664 A268E Y286F S322A A336G 14 54665 Y286F I291V S292A S322A15 54666 I13L S292A S322A A336R 16 54667 V76I I152V I291V A336R 17 54668I13L I152V I291V A336R 18 54669 V76I S292A S322A A336R 19 54670 P47FV76I I291V A336R 20 54671 V76I Y286F S322A A336R 21 54672 P47F Y286FI291V A336R 22 54673 V76I L88V S322A A336R 23 54674 V76I L88V S292AS322A A336G 24 54675 V76I Y286F I291V S322A A336R 25 54676 V53A A268EY286F I291V A336R 26 54677 V76I I152V Y286F S322A A336G 27 54678 I13LL88V Y286F I291V S292A 28 54679 I13L A268E Y286F I291V A336G 29 54680I13L V76I I152V S292A A336R 30 54681 I13L P47F V53A V76I I152V 31 54682P47F V76I Y286F I291V A336G 32 54683 P47F V53A I152V S322A A336G 2 54684— — — — — (JEA1)

TABLE 10 Measured Isobutanol Titers (from PNY2204, anaerobic passage #2)Variant Isobutanol identifier Titer (mM) 54654 5 54654 1 54654 7 5465550 54655 52 54655 57 54656 1 54656 2 54656 4 54657 51 54657 51 54657 5554658 2 54658 3 54658 1 54659 59 54659 58 54659 56 54660 33 54660 4354660 44 54661 32 54661 45 54661 28 54662 57 54662 58 54662 58 54663 3754663 33 54663 33 54664 43 54664 48 54664 48 54665 55 54665 51 54665 5954666 51 54666 56 54666 54 54667 42 54667 46 54667 42 54668 53 54668 5454668 55 54669 54 54669 38 54669 48 54670 2 54670 1 54670 2 54671 4554671 49 54671 58 54672 3 54672 2 54672 1 54673 32 54673 31 54673 2154674 32 54674 21 54674 34 54675 56 54675 62 54675 57 54676 57 54676 5754676 57 54677 50 54677 38 54677 46 54678 40 54678 43 54678 44 54679 2554679 39 54679 34 54680 43 54680 56 54680 48 54681 1 54681 2 54681 154682 4 54682 6 54682 3 54683 2 54683 2 54683 1 54684 41 54684 43 5468447

Example 2 Variants Generated and Tested in an Isobutanol ProductionPathway

TABLE 11 KARI Variants SEQ ID NO: Variant Identifier Amino AcidSubstitutions relative to JEA1) 6 54657 Y286F I291V A336R 11 54662 Y286FS322A I152V 2 54684 (JEA1) 33 65130 S322A I291V A336R 34 65131 Y286FS322A A336R 35 65132 Y286F S322A I291V 36 65133 Y286F S322A I291V A336R37 65134 S322A I152V A336R 38 65135 I291V I152V A336R 39 65136 S322AI291V I152V 40 65137 S322A I291V I152V A336R 41 65138 Y286F I152V A336R42 65139 Y286F S322A I152V A336R 43 65140 Y286F I291V I152V 44 65141Y286F I291V I152V A336R 45 65142 Y286F S322A I291V I152V 46 65143 Y286FS322A I291V I152V A336R 47 65144 Y286F S322A P47F I291V F50A A336R 4865145 S322A P47F I291V I152V F50A A336R 49 65146 Y286F S322A P47F I152VF50A A336R 50 65147 Y286F P47F I291V I152V F50A A336R 51 65148 Y286FS322A P47F I291V I152V F50A 52 65149 Y286F S322A P47F I291V I152V F50AA336R 53 65150 Y286F V76I S322A I291V I152V A336R 54 65151 Y286F V76IS322A I291V A68E A336R 55 65152 S322A I291V I152V I13L A336R 56 65153Y286F V53A S322A I291V A336R A268E 57 65154 Y286F V76I S322A I291V I152VI13L A336R 58 65155 I80T I291V I152V I13L A68E A336R 59 65156 Y286FV117I S322A I152V I13L 60 65157 Y286F V76I S322A I291V G72W A71K A336R61 65158 Y286F S322A I291V I152V I13L A336R A329E 62 65159 Y286F S86AS322A I80T I152V A68E 63 65160 Y286F S322A I291V I152V I13L F50N A336R64 65161 Y286F V76I S322A I291V G72W A71K A336R A268T

TABLE 12 Measured Isobutanol Titers (from PNY2238, anaerobic passage #2)Variant Isobutanol Isobutanol Isobutanol Identifier Titer (mM) Seq IDTiter (mM) Seq ID Titer (mM) 54657 53 65139 49 65150 86 54657 53 65139103 65151 54 54657 69 65139 100 65151 49 54662 61 65140 40 65151 8154662 63 65140 62 65151 91 54662 62 65140 75 65152 67 54662 80 65140 8265152 45 54684 52 65141 59 65152 98 54684 33 65141 50 65152 100 65130 5365141 58 65153 38 65130 61 65141 74 65153 53 65130 67 65142 53 65153 6265130 85 65142 59 65153 77 65131 55 65142 62 65154 47 65131 49 65142 7465154 66 65131 56 65143 51 65154 77 65131 100 65143 51 65154 60 65132 4465143 49 65155 47 65132 54 65143 101 65155 49 65132 55 65144 41 65155 5165132 85 65144 36 65155 85 65133 56 65144 47 65156 58 65133 63 65144 7665156 58 65133 87 65145 47 65156 67 65133 84 65145 67 65156 85 65134 6565145 94 65157 54 65134 58 65145 70 65157 47 65134 52 65146 55 65157 8765134 106 65146 46 65157 82 65135 61 65146 48 65158 56 65135 51 65146103 65158 44 65135 55 65147 57 65158 69 65135 81 65147 47 65158 69 6513670 65147 49 65159 53 65136 50 65147 86 65159 61 65136 82 65148 55 6515947 65136 87 65148 48 65159 65 65137 47 65148 69 65160 64 65137 40 6514863 65160 30 65137 63 65149 45 65160 39 65137 111 65149 47 65160 77 6513858 65149 55 65161 52 65138 50 65149 73 65161 50 65138 52 65150 44 6516149 65138 86 65150 82 65161 91 65139 28 65150 92

Example 3 Variants Generated and Tested in an Isobutanol ProductionPathway

TABLE 13 KARI Variants SEQ ID NO: Variant Identifier SubstitutionsRelative to JEA1 (SEQ ID NO: 2) 2 54684 (JEA1) 65 66824 I152V Y286FI291V S322A A336R 66 66825 R306K N267T I152V I291V S322A A336R 67 66826V76I V171I V156A I152V Y286F I291V S322A A336R 68 66827 S292L N114GM238I I152V Y286F I291V S322A A336R 69 66828 V76I S292L L88V I152V Y286FI291V S322A A336R 70 66829 V156A M238I A329E I152V Y286F I291V S322AA336R 71 66830 V156A I127V A68E I152V Y286F I291V S322A A336R 72 66831L88V K99N A277V I152V Y286F I291V S322A A336R 73 66832 V156A N114G L33CI152V Y286F I291V S322A A336R 74 66833 N267T F61L A68E I152V Y286F I291VS322A A336R 75 66834 V171I L33C Y286F I291V S322A A336R 76 66835 N114GI127V F61L I152V Y286F I291V S322A A336R 77 66836 S292L L88V A329V I152VY286F I291V S322A A336R 78 66837 V171I V156A I127V I152V Y286F I291VS322A A336R 79 66838 F61L A68E I152V I291V S322A A336R 80 66839 R275KI127V A329V I152V Y286F I291V S322A A336R 81 66840 I127V F61L A277VI152V Y286F I291V S322A A336R 82 66841 V76I R306K Y286F I291V S322AA336R 83 66842 V171I L33C I152V Y286F I291V A336R 84 66843 A68E I152VI291V A336R 85 66844 R275K A329E Y286F I291V S322A A336R 86 66845 F61LA329V I152V Y286F I291V A336R 87 66846 V152I N114G L88V I152V Y286FI291V S322A A336R 88 66847 V76I V156A A277V I152V Y286F I291V S322AA336R 89 66848 R275K I291V S322A A336R 90 66849 R275K K99N I152V Y286FI291V A336R 91 66850 V76I A329E A277V I152V Y286F I291V S322A A336R 9266851 V171I F61L I152V Y286F I291V A336R 93 66852 L33C A68E I152V I291VS322A A336R 94 66853 N114G A329E Y286F I291V S322A A336R 95 66854 R306KLK99N A329V I152V Y286F I291V S322A A336R 96 66855 N267T M238I L33CI152V Y286F I291V S322A A336R 97 66856 S292L L88V A277V I152V Y286FI291V S322A A336R 98 66857 S292L R275K L33C I152V Y286F I291V S322AA336R

TABLE 14 Measured Isobutanol Titers (from PNY2238, anaerobic passage #2)Variant Isobutanol Titer Identifier (mM) 54684 39 54684 38 54684 3066824 50 66824 37 66825 35 66825 31 66825 17 66826 33 66826 27 66826 2066827 42 66827 38 66828 28 66828 25 66829 44 66829 37 66829 31 66830 2566830 22 66830 18 66831 30 66831 23 66831 12 66832 6 66832 5 66832 466833 9 66833 8 66834 24 66834 18 66834 15 66835 24 66835 15 66836 3166836 30 66836 26 66837 33 66837 27 66837 26 66838 31 66838 27 66838 2166839 38 66839 28 66840 17 66840 16 66840 16 66841 41 66841 39 66841 3066842 37 66842 22 66842 22 66843 36 66843 31 66844 36 66844 35 66844 2766845 32 66845 25 66845 4 66846 37 66846 29 66847 31 66847 18 66847 1666848 42 66848 25 66848 21 66849 55 66849 38 66850 40 66850 36 66850 3066851 34 66851 32 66851 22 66852 20 66852 10 66853 36 66853 34 66853 3466854 34 66854 28 66855 10 66855 9 66856 33 66856 26 66856 17 66857 2466857 14 66857 14

Example 4 Variants Generated and Tested in an Isobutanol ProductionPathway

TABLE 15 KARI Variants SEQ ID Variant Substitutions Relative to VariantNO Identifier identifier 65138 (SEQ ID NO: 41) 41 65138 99 76377 A71K100 76378 G72W 101 76379 N114G 102 76380 M238I 103 76381 R306K 104 76382A329E 105 76383 A71K G72W 106 76384 A71K N114G 107 76385 A71K M238I 10876386 A71K R306K 109 76387 A71K A329E 110 76388 G72W N114G 111 76389G72W M238I 112 76390 G72W R306K 113 76391 G72W A329E 114 76392 N114GM238I 115 76393 N114G R306K 116 76394 N114G A329E 117 76395 M238I R306K118 76396 M238I A329E 119 76397 R306K A329E 120 76398 Y113F R280L 12176399 M238I E264K R280D 122 76400 Y239H F286W 123 76401 Y239H A329R 12476402 Y113F A329E 125 76403 R280H F286W 126 76404 A69K A71K 127 76405F286W A329E 128 76406 R306K S322A 129 76407 Q272T A329R 130 76408 A69KG72W 131 76409 N114S A295V 132 76410 G72W V152C 133 76411 N114S R280H134 76412 G72W I291N 135 76413 A69K K335M 136 76414 A71K R280L 137 76415A69T M301I 138 76416 A69T S322A 139 76417 E264K R280D A295V 140 76418N114G K335M 141 76419 S157T Y239H 142 76420 Q272T R306K 143 76421 R280LS322A 144 76422 N114G A329R 145 76423 N114S I291N 146 76424 E264K R280DM301I 147 76425 Y113F M301I 148 76426 R280H A295V 149 76427 V152C S157T150 76428 S157T I291N 151 76429 A69T N114G 152 76430 Q272T K335M 15376431 V152C M238I 154 76432 A69T A71K Y113F 155 76433 E264K R280D A329RK335M 156 76434 A69K V152C R280H 157 76435 N114S M301I A329E 158 76436Q272T F286W A295V 159 76437 G72W N114G Y239H 160 76438 R280L R306K S322A161 76439 S157T M238I I291N

TABLE 16 Measured Isobutanol Titers (from PNY2259) Cycle 1 Cycle 2Variant Isobutanol Isobutanol identifier Titer (mM) Titer (mM) 65138 2867 65138 34 57 65138 31 65 76377 22 60 76377 35 73 76377 27 59 76378 3158 76378 32 71 76378 36 63 76379 39 57 76379 32 59 76379 31 66 76380 1543 76380 30 68 76380 23 53 76381 18 31 76381 12 33 76381 9 23 76382 2757 76382 32 77 76382 17 44 76383 37 62 76383 29 59 76383 31 63 76384 3568 76384 36 66 76384 32 63 76385 36 71 76385 31 70 76385 30 62 76386 3658 76386 29 59 76386 19 41 76387 38 58 76387 54 73 76387 22 56 76388 3667 76388 20 49 76388 27 60 76389 29 54 76389 36 78 76389 28 83 76390 1146 76390 8 24 76390 13 37 76391 22 51 76391 25 54 76391 28 64 76392 3367 76392 41 67 76392 38 62 76393 3 4 76393 3 3 76393 5 7 76394 44 4776394 44 74 76394 24 52 76395 4 8 76395 6 6 76395 6 11 76396 28 70 7639626 60 76396 15 39 76397 13 40 76397 8 22 76397 6 15 76398 53 63 78398 2359 76398 28 64 76399 27 51 76399 31 58 76399 30 75 76400 5 9 76400 11 1476400 6 10 76401 48 63 76401 38 70 76401 48 65 76402 37 74 76402 32 6176402 31 84 76403 2 3 76403 3 10 76403 3 6 76404 6 9 76404 5 7 76404 4 976405 3 3 76405 6 8 76405 2 2 76406 36 61 76406 18 41 76406 29 56 7640728 57 76407 3 11 76407 35 65 76408 4 5 76408 2 4 76408 2 2 76409 36 5776409 50 62 76409 32 84 76410 18 49 76410 31 55 76410 25 55 76411 18 5676411 25 65 76411 17 46 76412 9 41 76412 15 37 76412 15 41 76413 2 376413 1 3 76413 3 5 76414 24 47 76414 37 58 76414 10 43 76415 47 8376415 40 70 76415 44 61 76416 30 65 76416 34 66 76416 26 68 76417 31 6176417 32 62 76417 20 52 76418 36 60 76418 65 71 76418 30 57 76419 33 6376419 29 57 76419 32 65 76420 15 40 76420 18 35 76420 18 45 76421 39 6476421 21 60 76421 47 65 76422 33 63 76422 37 62 76422 20 58 76423 1 276423 4 5 76423 2 3 76424 49 62 76424 39 62 76424 33 65 76425 48 7276425 42 62 76425 23 62 76426 24 62 76426 13 51 76426 21 50 76427 18 5176427 19 52 76427 25 52 76428 9 36 76428 16 34 76428 15 32 76429 31 6676429 36 56 76429 31 65 76430 30 50 76430 26 66 76430 35 69 76431 25 5576431 21 46 76431 22 57 76432 54 52 76432 40 75 76432 41 68 76433 47 5576433 38 52 76433 31 62 76434 3 4 76434 0 3 76434 4 7 76435 46 61 7643545 60 76435 35 56 76436 3 4 76436 5 5 76436 2 3 76437 53 82 76437 45 7876437 40 68 76438 17 48 76438 10 39 76438 14 46 76439 21 48 76439 17 4576439 17 38

Example 5 Variants for Isobutanol Production

Variants provided in Table 17 were generated and tested as described forExample 4, except the strain was PNY1556 (transformed with asingle-plasmid as described above), Anaerobic cycles 1 and 2 were 3 dayslong, Cycle 3 was 2 days long. Results are given in Table 18.

TABLE 17 KARI Variants Seq ID Variant Substitutions Relative to VariantNO: Number Identifier 65139 (Seq ID NO: 42) 42 65139 123 76401 Y239HA322S A329R 350 85023 Y239H 351 85024 A69T 352 85025 A71K 353 85026Y113F 354 85027 A69T Y239H 355 85028 A71K Y239H 356 85029 Y113F Y239H357 85030 A69T A71K 358 85031 A69T Y113F 359 85032 A71K Y113F 360 85033A69T A71K Y113F 361 85034 A71K Y113F Y239H 362 85035 A69T Y113F Y239H363 85036 A69T A71K Y239H 364 85037 A69T A71K Y113F Y239H 365 85038 A69TM238I Y239H 366 85039 A71K M238I Y239H 367 85040 Y113F M238I Y239H 36885041 A69T A71K M238I 369 85042 A69T Y113F M238I 370 85043 A71K Y113FM238I 371 85044 A69T Y239H M301I 372 85045 A71K Y239H M301I 373 85046Y113F Y239H M301I 374 85047 A69T A71K M301I 375 85048 A69T Y113F M301I376 85049 A71K Y113F M301I 377 85050 A69T Y239H A322S 378 85051 A71KY239H A322S 379 85052 Y113F Y239H A322S 380 85053 A69T A71K A322S 38185054 A69T Y113F A322S 382 85055 A71K Y113F A322S

TABLE 18 Measured Isobutanol Titers Isobutanol Titer (mM) Variant NumberCycle 1 Cycle 2 Cycle 3 65139 12 51 17 65139 5 49 30 65139 2 12 27 651392 13 34 76401 27 84 36 76401 12 70 48 76401 5 54 54 76401 2 9 18 8502321 67 19 85023 13 59 32 85023 12 44 43 85023 6 59 52 85024 10 55 9 850245 32 22 85024 4 35 40 85024 2 9 4 85025 5 49 24 85025 4 27 18 85025 4 2732 85025 1 3 2 85026 12 47 30 85026 3 9 5 85026 2 6 9 85026 2 12 3185027 18 83 29 85027 16 62 38 85027 13 53 44 85027 2 20 18 85028 25 60 385028 22 62 23 85028 9 64 18 85028 7 52 39 85029 12 48 33 85029 6 55 4385029 5 44 45 85029 3 49 58 85030 11 48 20 85030 10 51 23 85030 4 30 1985030 4 16 41 85031 12 49 17 85031 4 25 25 85031 4 29 40 85031 3 17 1685032 9 50 11 85032 5 29 21 85032 3 16 17 85032 1 8 15 85033 9 53 1385033 5 34 37 85033 4 20 8 85033 3 17 43 85034 18 65 22 85034 9 54 1385034 6 51 34 85034 6 48 29 85035 15 65 22 85035 13 55 27 85035 5 39 4585035 2 44 58 85036 20 58 17 85036 10 80 23 85036 6 48 54 85036 6 51 3785037 20 56 13 85037 6 52 53 85037 3 31 70 85037 3 13 11 — — — — — — — —— — — — — — — — 85038 27 81 37 85038 18 66 42 85038 16 68 35 85038 8 5240 85039 28 70 19 85039 28 78 25 85039 9 61 52 85039 4 28 40 85040 26 7736 85040 13 73 13 85040 10 62 30 85040 0 4 6 85041 21 60 23 85041 9 5822 85041 6 47 62 85041 2 15 10 85042 8 56 37 85042 8 45 38 85042 4 38 5485042 4 29 44 85043 18 62 22 85043 12 56 31 85043 10 60 40 85043 3 39 5485044 19 53 10 85044 12 54 25 85044 8 40 33 85044 2 23 43 85045 12 50 1885045 4 17 9 85045 3 14 32 85045 3 19 37 85046 6 44 21 85046 4 30 1885046 3 9 8 85046 2 6 8 85047 3 13 7 85047 3 16 8 85047 3 14 19 85047 211 12 85048 6 31 6 85048 1 4 4 85048 0 2 2 85048 0 9 55 85049 3 13 885049 2 6 4 85049 0 0 0 85049 0 0 0 85050 24 70 37 85050 21 68 27 8505020 70 21 85050 4 25 30 85051 24 65 36 85051 23 72 21 85051 6 65 40 850516 55 63 85052 27 77 24 85052 15 57 25 85052 10 59 36 85052 10 60 5285053 14 50 7 85053 8 49 22 85053 3 20 6 85053 0 20 44 85054 11 53 1985054 4 54 29 85054 1 6 2 85054 0 2 2 85055 4 10 1 85055 2 5 1 85055 1 42 85055 1 4 0

Example 6 Identification of Suitable Substitutions by Site-Saturationfor Residues 152, 286, 336

All 20 amino acids at sites 152, 286, and 336 of parent variantR8—SOG1_y2 (amino acid SEQ ID NO: 346; nucleic acid SEQ ID NO: 383) wereindividually constructed and tested. This parent variant is JEA1 withsubstitutions A68E, I152V, Y286F, and A336R.

The General Methods are the same as in the application for Examples 1-4.The two-plasmid system was used, with KART on a pLH556-based plasmid,and DHAD on pBP915. The yeast strain was PNY2259.

Site-Directed Mutagenesis and Subcloning into pLH556-Based Vector:

Position 152 was mutagenized by overlap-extension PCR with mutagenicprimers. The KARI inserts were subcloned into pLH556 (with the KARIremoved) by yeast gap-repair cloning. The sequence of the KARI portionand flanking regions of the resulting plasmids was confirmed by DNAsequencing (Sanger method, ABI Prism 3730xl DNA Analyzer),

Positions 286 and 336 were mutagenized using the QuikChangeII kit(Agilent); position 286 in a pCR2.1 vector (invitrogen) and position 336in a TOPO Blunt vector (Invitrogen). The KARI variants were sequenced inthe Invitrogen vectors, then subcloned by standard ligation into thePmeI and SfiI restriction sites of pLH556. The KARI inserts wereconfirmed by sequencing again in the pLH556 vector.

The resulting 60 variants are characterized in the table below as eitherfunctional (F, defined as supporting a mean yeast isobutanol productionof at least 10% of the mean of the parent, or non-functional (NF,defined as not supporting a mean yeast isobutanol production of at least10% of the mean of the parent).

Quantitative data (percentages of parental isobutanol titer) areprovided in the second table. Data are based on the first anaerobicpassaging cycle.

TABLE 19 Substitutions Amino Acid Position 152 (F/NF) Position 286(F/NF) Position 336 (F/NF) A, Ala F F F C, Cys F F F D, Asp F NF F E,Glu F NF F F, Phe F F F G, Gly F NF F H, His F F F I, Ile F F F K, Lys FNF F L, Leu F NF F M, Met F F F N, Asn F NF F P, Pro F NF F Q, Gln F NFF R, Arg F NF F S, Ser F NF F T, Thr F NF F V, Val F NF F W, Trp F F FY, Tyr F F F

TABLE 20 Quantitative Data for the substitutions in Table 24 (expressedas a percentage of the parent's mean isobutanol titer) - First AnaerobicCycle % of Parental % of Parental % of Parental Isobutanol IsobutanolIsobutanol Titer, Position Titer, Position Titer, Position Amino Acid152 286 336 A, Ala 51% 16% 106% C, Cys 128% 15% 98% D, Asp 54% 1% 112%E, Glu 77% 1% 89% F, Phe 67% 100% 114% G, Gly 25% 1% 95% H, His 90% 38%121% I, Ile 46% 30% 89% K, Lys 61% 2% 126% L, Leu 52% 10% 103% M, Met53% 40% 94% N, Asn 57% 1% 87% P, Pro 34% 1% 76% Q, Gln 82% 5% 131% R,Arg 42% 3% 100% S, Ser 86% 6% 115% T, Thr 89% 3% 100% V, Val 100% 9% 96%W, Trp 82% 17% 100% Y, Tyr 46% 51% 132%

What is claimed is:
 1. A polypeptide having ketol-acid reductoisomeraseactivity, wherein the polypeptide comprises a. the amino acid sequenceof SEQ ID NO: 41; b. at least 99% identity to SEQ ID NO: 41; c. at least99% identity to SEQ ID NO: 41 and at least one of the followingsubstitutions: M301I, Y239H, Y113F, S322A, A71K, N114G, A329R, A69T,N114S, G72W, A295V, E264K, R280D, A329E, S157T, M238I, Q272T, K335M,R280H, and a combination thereof; or d. an active fragment of any one of(a) to (c).
 2. The polypeptide of claim 1, further comprising asubstitution at at least one of position L33, P47, F50, F61, I80, andV156.
 3. The polypeptide of claim 1 further comprising at least one ofthe following substitutions: I13L, P47Y, F50A, V53A, S86A, A268E, V76I,L88V, and a combination thereof.
 4. A recombinant microbial host cellcomprising the polypeptide of claim
 1. 5. The recombinant microbial hostcell of claim 4 wherein said host cell further comprises reduced oreliminated acetolactate reductase activity.
 6. The recombinant microbialhost cell of claim 5 wherein said host cell further comprises at leastone deletion, mutation, and/or substitution in fra2.
 7. The recombinantmicrobial host cell of claim 4 further comprising the substrate toproduct conversions: a. pyruvate to acetolactate b.2,3-dihydroxyisovalerate to α-ketoisovalerate c. α-ketoisovalerate toisobutyraldehyde; and d. isobutyraldehyde to isobutanol.
 8. Therecombinant microbial host cell of claim 4 wherein said host cell is ayeast host cell.
 9. The recombinant microbial host cell of claim 8wherein said host cell is Saccharomyces cerevisiae.
 10. A method forconverting acetolactate to dihydroxyisovalerate comprising: a. providinga microbial host cell comprising a polypeptide of claim 1; b. contactingthe polypeptide with acetolactate wherein dihydroxyisovalerate isproduced.
 11. A method of producing a product selected from the groupconsisting of isobutanol, pantothenate, valine, leucine, isoleucine, 3,3-dimethylmalate, and 2-methyl- 1-butanol or combinations thereofcomprising: a. providing the recombinant host cell of claim 4 whereinthe recombinant host cell comprises a product biosynthetic pathway; andb. contacting the microbial host cell with a carbon substrate underconditions whereby the product is produced.
 12. The method of claim 11wherein at least a portion of the contacting occurs under anaerobicconditions.